LAPORAN AWAL PRAKTIKUM

KIMIA BAHAN ALAM FARMASI

ISOLASI SENYAWA TRITERPENOID DARI PEGAGAN
(Centella asiatica L.)

OLEH:

NAMA : ALFATHRI YUNEDI

NO BP : 1811013018
SHIFT /KLP : 1 (SENIN) / 4
HARI/TGL : SELASA/12 MEI 2020
REKAN KERJA : 1. MAISUNNA BUNGA ULI 1811011046
2. SUCI WULANSARI 1811012040
3. YULMA HERDALINA 1811012052

LABORATORIUM KIMIA BAHAN ALAM FARMASI

FAKULTAS FARMASI
UNIVERSITAS ANDALAS
PADANG
2020
 BAB I
PENDAHULUAN
1.1 Tujuan
1. Mengetahui dan mempraktekkan cara mengisolasi triterpenoid.

2. Mengetahui cara mengidentifikasi triterpenoid.

1.2 Manfaat
1.Dapat mengetahui cara mengisolasi senyawa triterpenoid.

2.Dapat mengetahui senyawa aktif yang berkhasiat sebagai obat didalam

tumbuhan pegagan.

3.Dapat mengisolasi senyawa asiatikosida, asam madekasat dan asam asiatat dari
tumbuhan Centella asiatica L.
 BAB II
TINJAUAN PUSTAKA
2.1 Tinjauan Botani

Gambar 1. Tumbuhan pegagan (Centella asiatica L. Urban) 1

2.1.1 Klasifikasi
Berdasarkan klasifikasi taksonomi, pegagan termasuk ke dalam
Divisi : Spermatophyta.
Subdivisi : Angiospermae,
Kelas : Dicotyledonae,
Ordo : Umbillales,
Famili : Umbilliferae (Apiaceae),
Genus : Centella,
Spesies : Centella asiatica (L.) Urban atau Hidrocotyle asiatica Linn 1

2.1.2 Morfologi
Pegagan (Centella asiatica (L.) Urban.) merupakan tanaman herba tahunan yang
tumbuh di daerah tropis dan berbunga sepanjang tahun. Bentuk daunnya bulat seperti
ginjal manusia, batangnya lunak dan beruas, serta menjalar hingga mencapai satu
meter. Pada tiap ruas tumbuh akar dan daun dengan tangkai daun panjang Pegagan
(Centella asiatica (L.) Urban.) merupakan tanaman herba tahunan yang tumbuh di
daerah tropis dan berbunga sepanjang tahun. Bentuk daunnya bulat seperti ginjal
manusia, batangnya lunak dan beruas, serta menjalar hingga mencapai satu meter. Pada
tiap ruas tumbuh akar dan daun dengan tangkai daun panjang sekitar 5– 15 cm dan akar
berwarna putih, dengan rimpang pendek dan stolon yang merayap dengan panjang 10–
80 cm. Tinggi tanaman berkisar antara 5,39–13,3 cm, dengan jumlah daun berkisar
antara 5–8,7 untuk tanaman induk dan 2–5 daun pada anakannya.2
Bunga umumnya 3, yang ditengah duduk, yang disamping bertangkai pendek,
daun pelindung 2, panjang 3-4 mm, bentuk bulat telur, mahkota bunga berwarna merah
lembayung, panjang 1-1,5 mm, lebar sampai 0,75 mm. buah pipih lebar lebih kurang 7
mm dan tinggi lebih kurang 3mm, bertekuk dua, jelas berusuk, berwarna kuning
kecoklatan, berdinding agak tebal.2
Helai daun tunggal, berbentuk ginjal, dan bertangkai dengan panjang berkisar 5-
15 cm. Tepi daun bergerigi tersusun dalam roset yang terdiri dari 2-10 helai daun.
Bunganya berwarna putih atau merah muda, tunggal atau sejumlah 3-5 bunga muncul
bersamaan dari ketiak daun. Buah pegagan ini berukuran kecil, berbentk lonjong atau
pipih dengan panjang 2-2,5 mm, beraroma wangi, dan kerasa pahit, bagian yang
digunakan sebagai bahan herbal adalah seluruh bagian tanaman pegagan.3

2.1.3 Habitat dan distribusi

Tanaman pegagan mudah tumbuh dan mempunyai daya adaptasi yang luas .
Pegagan tumbuh baik pada tanah yang agak lembap, tetapi cukup sinar matahari atau
agak terlindung. Pegagan tumbuh optimun di dataran medium pada ketinggian sekitar
700 m dpl, namun juga mampu tumbuh di daerah tinggi hingga 2.500 m dpl. Secara
empiris tanaman pegagan mempunyai syarat tumbuh spesifik dalam hal kebutuhan
cahaya matahari, yang akan memengaruhi bentuk morfologi daun dan kandungan
bioaktif.4
Pegagan merupakan tumbuhan tropis dengan daerah penyebaran cukup luas, dari
dataran rendah sampai dataran tinggi, hingga 2.500 m di atas permukaan laut. Pegagan
dapat ditemukan di daerah perkebunan, ladang, tepi jalan, pematang sawah, ataupun di
ladang yang agak basah.4

2.2 Kandungan Kimia

 Salah satu metabolit sekunder yang terkandung dalam tumbuhan pegagan adalah
triterpen. Triterpen merupakan senyawa yang banyak terdapat dalam tumbuhan.
Triterpena termasuk ke dalam kelompok senyawa terpenoid. Kata terpena mencakup
sejumlah besar senyawa tumbuhan dan istilah ini digunakan untuk menunjukkan
bahwa secara brosintesis semua senyawa tumbuhan itu berasal dari senyawa yang
sama, yaitu dari molekul isopren.4
Pegagan memiliki kandungan kimia seperti saponin, triterpen (asiatikosida,
brahmosida, thankunusida dan alkaloid (hidrokofilin), isothankusida, madekusosida,
brahmasida, asam brahmik, asam mudasiatik. Selain itum juga terdapat meso-inisitol,
sentelloso, nasetonoid, garam K, Ne, Cn, Fe, velasin, tasin, musilago, resin, pectin, gula
dan vitamin B.6

Gambar 2 Stuktur Asiatikosida

Gambar 3 Stuktur Asam Madekasat

2.3 Kegunaan Tradisional

 Pegagan (Centella asiatica (L) Urban.) mengandung sejumlah besar senyawa golongan
saponin triterpenoid pentasiklik. Pegagan diketahui mempunyai aktivitas farmakologis yang
luas yaitu untuk menyembuhkan luka terbuka, untuk mengobati berbagai kondisi kulit seperti
kusta, lupus, varises, bisul, eksim, diare, demam. Suatu penyakit pada saluran genitourinary
perempuan dan jika untuk menghilangkan cemas dan meningkatkan daya ingat, antibakteri,
antioksidan, antifungi, antiinflamasi.10
Pegagan (Centella asiatica (L.) Urban.) mempunyai banyak manfaat. Pegagan
(Centella asiatica (L.) Urban.) secara tradisional banyak digunakan untuk mengobati penyakit
kulit. Pegagan (Centella asiatica (L.) Urban.) juga dapat digunakan untuk mengobati sakit
perut, batuk, batuk berdarah dan disentri, penyembuh luka, radang, pegal linu, asma, wasir,
tuberculosis, lepra, demam, dan penambah selera makan.2
Sejak dahulu, daun pegagan telah digunakan secara turun-temurun untuk
mengobati penyakit kulit, mengatasi gangguan saraf, dan memperbaiki peredaran darah.
Bahkan, daun pegagan kerap dikonsumsi sebagai lalapan oelh masyarakat di Jawa Barat.
Seiring dengan perkembangan teknologi, daun pegagan kemudian di ekstraksi dengan
perkembangan teknologi, daun pegagan kemudian di ekstrak dan diolah dalam bentuk teh,
kapsul krim, salep, obat jerawat dan body lotion. 2
2.4 Bioaktivitas
2.4.1 Ektrak
Ektrak methanol pegagan memiliki bioktivitas sebagai antibakteri pada
pemberian konsentrasi 80% dan 100% dimana pada pengamatan minggu pertama dan
minggu kedua menunjukkan tidak adanya tanda-tanda pertumbuhan koloni bakteri
yang ditandai dengan jumlah koloni 0%. 4
Hasil penelitian Alvonsus (2010) mendapatkan bahwa ektrak haerba pegagan
memilik daya antiinflamasi dengan nilai IC50 sebesar 158,79 ug/ml dengan kisaran
97,21 ug/ml-191,55 ug/ml.5
2.4.2 Metabolit Sekunder
1. Asam asiatik
 Asam asiatik memiliki efek perlindungan antiinflamasi pada sel HUVE dan tikus
diabetes pada hewan perawatan induvidu. Kombinasi asam Asiatic dan carnosine
menghasilkan lebih banyak pengurangan dalam fragmentasi DNA dari pada perawatan
carnosine atau asamasitik saja(P<0.05) penurunan kadar sitokin inflamasi dan ROS
(P<0,05), penurunan produksi TNF-alpha dan ROS dari pada pengobatan carnosine
atau AA saja (P <0,05). Perawatan Carnosine atau AA pada 1 Μm menurunkan
aktivitas mengikat ini (P <0,05).6
Asam asiatik juga dapat menghambat respon inflamasi yang di induksi LPS pada
gingiva manusia fibrolast, asam asiatik secara signifikan menghambat produksi PGE2,
NO, TNF-α, IL-6, dan IL-8 yang diinduksi LPS baik dalam vivo dan in vitro. Hasil ini
menunjukkan bahwa asam asiatik memiliki kemampuan untuk menipiskan peradangan
yang diinduksi LPS pada HGF.7
2. Asiatikosida
Asiatikosida berkhasiat meningkatkan vitalitas dan daya ingat serta mengatasi
pikun yang berkaitan erat dengan asam nukleat. Glikosida dan triterpenoid adalah
triterpenoid asiatikosida turunan ἀ-amirin.3.Asiatikosida merupakan bagian dari
triterpenoid yang berfungsi menguatkan sel-sel kulit dan meningkatkan perbaikannya,
menstimulasi sel darah dan sistemimun, dan sebagai antibiotic alami. Senyawa bioaktif
asiaticoside dapat mempercepat proses penyembuhan luka dan bermanfaat untuk kusta
danpengobatan tuberculosis.8

2.5 Metode Ektraksi

Metode ektraksi yang dipilih adalah metode yang cocok dengan sifat bahan alam
yang digunkan. Penggunaan pelarut yang ideal untuk mengekstraksi adalah pelarut
yang menunjukkan selektivitas maksimal, mempunyai kapasitas terbaik, dan
kompatibel dengan sifat bahan yang diekstraksi. 9
Ekstraksi herba pegagan dilakukan dengan metode maserasi menggunakan
pelarut etanol 70%. Pemilihan metode ini karena metode ini mudah, menghasilkan
rendemen yang tinggi serta meminimalisir kerusakan senyawa kimia karena maserasi
tidak disertai panas. Walaupun menghasilkan rendemen yang lebih kecil dibandingkan
metode lain.10
Pelarut yang digunakan adalah etanol dengan konsentrasi 70% dimana
ekstraksinya dapat menyari zat aktif berupa asiatikosida paling banyak dibandingkan
pelarut etanol 30% dan 50%. Tidak menggunakan pelarut etanol 90% karena akan
banyak melarutkan klorofil sehingga ekstrak akan sangat lengket dan sulit
dikeringkan.10
Proses maserasi kemudian dilanjutkan dengan pembuatan ekstrak kental dimana
hasil maserasi dikumpulkan lalu diuapkan menggunakan waterbath.10
 BAB III
PROSEDUR KERJA
3.1 Alat dan Bahan
3.1.1 Alat
Wadah untuk maserasi, kolom kromatografi, corong, botol 100 mL, vial, pipet
tetes, seperangkat alat rotary evaporator, chamber, penotol.
3.1.2 Bahan
Daun pegagan kering (100 g), metanol, etil asetat, plat KLT, kapas, norit,
penampak noda untuk triterpen.

3.2 Cara Kerja

Daun Pegagan

Dikeringkan

Daun Pegagan Kering

Digrinder

Serbuk Pegagan (200 g)

Meserasi metanol 500 ml
Selama 3 hari
disaring

Maserat
Di beri norit

Filtrat
Rotary Evaporator

Ekstrak Kental
Rekristalisasi dengan Etil Asetat dan
metanol
Kristal Asiatikosida
Uji Klt dan Hitung Rf
Didapatkan Rf dari
Asiatikosida
 DAFTAR PUSTAKA

1. Yohanes, S. Kandungan Bahan Aktif Tanaman Pegagan Dan Khasiatnya Untuk

Meningkatkan Sistem Imun Tubuh Bioactive Compounds In Pegagan Plant And Its
Use For Increasing Immune System. Jurnal Litbang Pertan. 2016; 35:121–130.
2. Rohmawati, M. Karakteristik Morfologi Dan Anatomi Pegagan (Centella Asiatica
(L) Urban) Di Kabupaten Batang. Skripsi (2015).
3. Herlina, F. Diabetes Kandas Berkat Herbal. Jakarta : FM Media; 2013
4. Howan, Dian HO. Isolasi dan Identifikasi Metabolit Sekunder dari Ekstrak Butanol
Pegagan (Centella asiatica (L) urban). Journal of Chemistry . 2017; vol 2. No 2: 92-
95.
5. Yan, S., Wang, Z., Mong, M., Yang, Y. & Yin, M. Combination Of Carnosine And
Asiatic Acid Provided Greater Anti- Inflammatory Protection For Huve Cells And
Diabetic Mice Than Individual Treatments Of Carnosine Or Asiatic Acid Alone.
Food Chem. Toxicol. 2019: 1(2): 192–198.
6. Hao, C. Et Al. International Immunopharmacology Asiatic Acid Inhibits Lps-
Induced In Fl Ammatory Response In Human Gingival Fi Broblasts. Int.
Immunopharmacol. 2017; 6(3): 313–318.
7. Vinolina, N. S., Nainggolan, M. & Siregar, R. Journal Of Agricultural Science
Production Enhancement Technology Of Pegagan. Journal Of Agricultural. 2018;
4(2): 304–312.
8. Herlinda, D. & Howan, O. Isolasi dan identifikasi metabolit sekunder dari ekstrak
butanol pegagan [ Centella asiatica ( L ) urban ]. Jurnal Saintek. 2017; 1(2): 92–95.
9. Mukhriani. Ekstraksi, pemisahan senyawa, dan identifikasi senyawa aktif. J.
Kesehat. 2014; 1(7): 361–367.
10.Hapsari, widarika, S., Rohmayanti, Fitriana, Y., Et all. Skrining Fitokimia Ekstrak
Etanol Herba Pegagan dan Analisa Rendemen. Journal Of University Research
Collaquium : 2017.
 REVIEW JURNAL

Judul : Depigmented Centella asiatica Extraction by Pretreated with

Supercritical Carbon Dioxide Fluid for Wound Healing
Application
Nama Jurnal : MDPI
Volume & halaman : Processes 2020, 8, 277; doi:10.3390/pr8030277
Tahun : 2020
Penulis : Warintorn Ruksiriwanich , Chiranan Khantham , Korawan
Sringarm , Sarana Sommano and Pensak Jantrawut
Reviewer : Alfathri Yunedi (1811013018)
Metode Penelitian :
Persiapan Sampel Centella asiatica
Daun C. asiatica dikumpulkan dari Nampu, Ratchaburi, Thailand, selama bulan Januari sampai
Juni 2018, oleh ahli botani dari Penelitian dan Pengembangan Produk Farmasi dan Alam Unit
(PNPRDU), Universitas Chiang Mai, Chiang Mai, Thailand. Spesimen voucher herbarium
(PNPRDU004 / 62) disimpan di PNPRDU, Fakultas Farmasi, Universitas Chiang Mai. Daun
kering digiling menjadi potongan-potongan kecil dan disimpan pada suhu 4 ◦C sampai
digunakan.

Metode Ekstraksi
Ekstraksi Konvensional
Untuk persiapan ekstrak konvensional, bubuk C. asiatica (2 kg) dimaserasi dengan 70% etanol
(1: 4,5, b / b) pada suhu kamar selama 48 jam, mengikuti protokol ekstraksi Nasional Thailand
Daftar Obat Esensial [16]. Kemudian, ekstrak disaring melalui kertas Whatman No. 1, dan
filtrat diuapkan pada suhu 50 ◦C untuk menghilangkan pelarut menggunakan rotary evaporator
(Hei-VAP Precision, Heidolph, Schwabach, Jerman) sampai benar-benar kering. Sampel itu
bernama CV.
Pretreatment Menggunakan scCO2
Untuk pretreatment menggunakan ekstraksi scCO2, bubuk C. asiatica (3,6 kg) ditempatkan ke
dalam ekstraktor fluida superkritis (Guangzhou Gongcheng Teknologi Ilmu Digital,
Guangzhou, Cina) pada 35 MPa, 60 ◦C selama 3 jam, dengan 95% etanol (2 kg) sebagai
cosolvent. Volume ekstraktor adalah 24 L. Rasio etanol terhadap CO2 adalah 1:25 berat. Proses
ini menghilangkan pigmen warna dari C. asiatica powder. Kemudian, bubuk olahan C. asiatica
dimaserasi dengan etanol 85% (1: 4,5, b / b) pada suhu kamar selama 24 dan 48 jam untuk
mendapatkan ekstrak scCO2 1 (SCE1) dan 2 (SCE2), masing-masing. Etanol 85% digunakan
dalam proses maserasi, karena memberikan triterpen total tertinggi dan triterpenoid glikosida
dalam penelitian pendahuluan kami. Setiap ekstrak disaring dan diuapkan hingga kering

Identifikasi Senyawa Aktif dengan Kromatografi Lapis Tipis Kinerja Tinggi (HPTLC)
Persiapan Ekstrak Mentah dan Solusi Standar
Ekstrak C. asiatica (CV, SCE1 dan SCE2) didispersikan dalam 5 mL metanol pada 160 μL /
mL. Setiap sampel disonikasi selama 10 menit dan disentrifugasi untuk mengumpulkan
supernatan. Asiaticoside Standar rujukan Farmakope Amerika Serikat (USP RS) dilarutkan
dalam metanol pada 0,5 mg / mL untuk larutan standar A. USP RS ekstrak bubuk C. asiatica
didispersikan dalam metanol di 10 mg / mL, disonikasi selama 10 menit dan disentrifugasi.
Supernatan dikumpulkan dan digunakan sebagai standar solusi B
HPTLC
Sistem kromatografi lapis tipis (HPTLC) kinerja tinggi Camag (Muttenz, Swiss) dilengkapi
dengan sampler kromatografi lapis tipis otomatis (KLT) ATS 4, lapisan tipis pemindai
kromatografi (TLC) 3 dan perangkat lunak terintegrasi winCATS versi 1.2.3 digunakan untuk
analisis. HPTLC dilakukan pada pelat HPTLC (20 cm × 10 cm) yang diendapkan dengan silika
gel 60F254 (ketebalan lapisan 0,20 mm).

Penentuan Senyawa Aktif dengan Kromatografi Cair Kinerja Tinggi (HPLC)

Persiapan Ekstrak Mentah dan Solusi Standar
Menurut USP 40-NF 35, asiaticoside USP RS dilarutkan dalam metanol pada 0,05 mg / mL
(solusi standar A). USP RS ekstrak bubuk C. asiatica didispersikan dan disonikasi dalam
metanol pada 5,0 mg / mL (larutan standar B). 1 g ekstrak C. asiatica (CV dan SCE1) dan
methanol (100 mL) dipindahkan ke alat Soxhlet (GLASSCO, Ambala, India), diekstraksi
selama 8 jam, dan diencerkan dengan metanol hingga 100 mL (larutan stok sampel). Solusi
sampel disiapkan oleh larutan stok sampel encer (5 mL) dengan metanol hingga 10 mL.
Sebelum injeksi, solusinya adalah disaring (ukuran pori 0,45 μm), membuang beberapa
mililiter pertama dari filtrat
HPLC
Total triterpenoid dan glikosida triterpenoid total dalam ekstrak standar dan C. asiatica
ditentukan dengan kromatografi cair kinerja tinggi (HPLC) menggunakan kolom Luna® C18
(250) mm × 4,60 mm, 5 μm; Fenomeneks, Torrance, CA, AS), detektor LC1200UV / VIS dan
LC1100HPLC pompa. Pemisahan kromatografi dicapai menggunakan sistem fase gerak biner
0,3% (v / v) asam fosfat dan asetonitril di bawah gradien elusi, sesuai dengan komposisi
turunan triterpene C. asiatica dalam monografi USP 40-NF35. Volume injeksi dan laju aliran
masing-masing, 10 μL dan 1 mL / menit, dan detektor UV ditetapkan pada 200 nm.

Skrining Antioksidan oleh DPPH Radical Scavenging Activity

Aktivitas pembersihan radikal DPPH dari CV dan SCE1 ditentukan menggunakan sebelumnya
metode yang dijelaskan [17] dengan sedikit modifikasi. Secara singkat, lima konsentrasi encer
masing-masing sampel, bersama dengan 50 μL air suling dan 50 μL larutan DPPH etanol 0,1
mM, adalah ditambahkan ke sumur dari lempeng 96-sumur (Nalge Nunc International,
Rochester, NY, USA). Reaksinya campuran diinkubasi pada 25 ± 2 ◦C selama 30 menit,
kemudian solusinya dipisahkan, dipindahkan ke sumur-96 lempeng mikro, dan absorbansi
diukur pada 515 nm terhadap kosong (etanol) menggunakan pembaca plat (EZ Baca 400 Flexi,
Biochrom, Cambridge, UK). Standar asam askorbat adalah kontrol positif. Semua percobaan
dilakukan dalam rangkap tiga.

Hasil Penelitian
Identifikasi Senyawa Bioaktif oleh HPTLC
Dalam penelitian ini, C. asiatica ditangani menggunakan ekstraksi scCO2 dengan etanol
sebagai cosolvent oleh maserasi etanol selama 24 dan 48 jam untuk mendapatkan SCE1 dan
SCE2, masing-masing. Beberapa laporan menunjukkan bahwa ekstraksi etanol oleh maserasi
menghasilkan jenis senyawa biologis aktif yang sama,
tetapi dalam jumlah yang berbeda relatif terhadap pretreatment menggunakan ekstraksi scCO2
[34-37]. Di sini, SCE1 tadinya dipilih untuk analisis lebih lanjut karena hasilnya yang lebih
besar daripada SCE2, dan profil senyawa yang sama antara SCE1 dan SCE2 ditemukan dalam
kromatogram HPTLC.

Aktivitas Biologis Ekstrak Centella asiatica

Menunjukkan aktivitas biologis (aktivitas DPPH dan ABTS + radikal)
lebih tinggi untuk CV (46,85 ± 2,31% dan 0,017 ± 0,004 mg TEAC / g) daripada SCE1, yang
mungkin disebabkan oleh daya kelarutan tinggi scCO2, yang diterapkan 3 jam sebelum proses
maserasi etanol dalam ekstraksi C. asiatica (SCE1). Kurva standar Trolox di ABTS +
scavenging assay adalah
y = −7.547x + 1.4813, r
2 = 0,995.
Selain itu, total konten fenolik lebih besar untuk CV (1,391 ± 0,044 μg setara asam galat
[GAE] / g ekstrak) dari SCE1 (0,953 ± 0,031 μg GAE / g ekstrak). Metode ekstraksi scCO2
dengan etanol sebagai cosolvent bisa memfasilitasi pembubaran senyawa polar, seperti yang
mengandung gugus fenolik, dalam ekstrak SCE1, menyebabkan kandungan fenolik total yang
relatif lebih rendah, dan aktivitas antioksidan (DPPH dan ABTS +). Trisaccharides, yaitu
asiaticoside dan madecassoside, memiliki polaritas yang lebih tinggi daripada aglikon, yang
meliputi asam madecassic dan asam asiatik, yang menyebabkan kelimpahan yang lebih rendah
dari senyawa-senyawa ini dalam SCE1 dari pada CV

Kesimpulan
Pretreatment C. asiatica menggunakan ekstraksi scCO2 dengan etanol sebagai cosolvent
sebelum etanol maserasi dapat menghilangkan pigmen tanaman, menghindari pemudaran
warna dan variasi warna dalam produk, dan memberikan ekstrak dengan aktivitas
penyembuhan luka in vitro yang lebih besar daripada metode konvensional.
 processes
Article
Depigmented Centella asiatica Extraction by
Pretreated with Supercritical Carbon Dioxide Fluid
for Wound Healing Application
Warintorn Ruksiriwanich 1,2, * , Chiranan Khantham 2 , Korawan Sringarm 1,3 ,
Sarana Sommano 1,4 and Pensak Jantrawut 1,2
1 Cluster of Research and Development of Pharmaceutical and Natural Products Innovation for Human or
Animal, Chiang Mai University, Chiang Mai 50200, Thailand; korawan.s@cmu.ac.th (K.S.);
sarana.s@cmu.ac.th (S.S.); pensak.j@cmu.ac.th (P.J.)
2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200,
Thailand; ckhantham@gmail.com
3 Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai
50200, Thailand
4 Plant Bioactive Compound Laboratory, Department of Plant and Soil Sciences, Faculty of Agriculture,
Chiang Mai University, Chiang Mai 50200, Thailand
* Correspondence: warintorn.ruksiri@cmu.ac.th; Tel.: +66-53-944338




Received: 6 February 2020; Accepted: 24 February 2020; Published: 28 February 2020


Abstract: Centella asiatica has been included in Thai traditional medicinal plants and recipes, as
a well-established historical use as a vegetable and tonic. However, when applied in modern
formulations, the progressive degradation of the plant pigments occurs, causing color-fading and
color variation in the products. Depigmentation of the comminuted sample using supercritical carbon
dioxide (scCO2 ) fluid extraction with a cosolvent was introduced as a pretreatment to solve the
color-fading problem. The contents of compounds with known biological activities and the wound
healing activities (antioxidant screening by DPPH and ABTS+ scavenging activities; cell migration
assay; matrix metallopeptidase [MMP]-2 inhibition on human skin fibroblast; endothelial cell tube
formation assay) of the C. asiatica leaf extracts obtained by conventional ethanolic extraction (CV)
and pretreatment using scCO2 extraction, were determined. Total triterpenoids (madecassoside,
asiaticoside B, asiaticoside, madecassic acid, terminolic acid and asiatic acid) and total triterpenoid
glucosides (madecassoside, asiaticoside B and asiaticoside) were notably more abundant in the
extract that had been pretreated using scCO2 than the extract obtained by CV. Moreover, the scCO2
pretreatment not only caused greater relative MMP-2 inhibition (58.48 ± 7.50% of the control), but
also exhibited a higher cell migration (59.83 ± 1.85% of the initial) and number of vessels (18.25 ±
4.58) of angiogenesis in the wound healing process. Additionally, positive correlations were observed
between the DPPH antioxidant activity and madecassoside content (r = 0.914, p < 0.01), as well as
between the cell migration activity and asiaticoside content (r = 0.854, p < 0.05). It can be concluded
that the scCO2 pretreatment of C. asiatica can eliminate color pigments from the extract and improve
its in vitro wound healing activity.

Keywords: angiogenesis; cell migration; Centella asiatica; depigmentation; supercritical carbon dioxide
fluid extraction; wound healing

1. Introduction
Wound healing is critical in maintaining the physical barrier function of the skin. It plays a
pivotal role to protect the body from pathogens when the skin is damaged. The wound healing

Processes 2020, 8, 277; doi:10.3390/pr8030277 www.mdpi.com/journal/processes

Processes 2020, 8, 277 2 of 15

process involves three overlapping stages: inflammation, proliferation and remodeling [1]. During the
inflammatory stage, edema and erythema result from the activation of inflammatory cytokines and the
action of the immune cells, which are necessary for angiogenesis and fibroplasia. Cell migration occurs
to protect against environmental pathogens, and local hemorrhaging is stopped by coagulation factors.
In the second or proliferation stage, cells contract to close the wound, and angiogenesis is initiated,
forming new blood vessels to deliver nutrients, fluid and oxygen to the cells in the wound area. Finally,
the remodeling stage is the last recovery process. The stimulation of collagen fiber production in the
cells is required to remodel the tissue for full recovery [2].
It has been evident for several years that matrix metallopeptidases (MMPs) are expressed
throughout the wound healing process, although the mechanisms through which these proteinases
function are only now being discovered. To date, it appears that almost all MMPs are positive regulators
of these stages. However, it has been suggested that both MMP-2 and MMP-9 may have inhibitory
effects upon cell proliferation [3], and so natural sources of bioactive compounds that can inhibit
MMP-2 activity provide positive feedback regulation to induce cell proliferation.
Centella asiatica is a famous Thai traditional herb. In Thailand, C. asiatica aqueous leaf extract
is popular as an everyday beverage because of its longevity effect. Several biological activities of
the C. asiatica ethanolic extract have been reported, such as in vitro anticancer [4], antioxidant [5] and
antimicrobial [6], besides in vitro and in vivo wound healing [7,8]. The C. asiatica leaf extract contains
four triterpenes, including madecassoside, asiaticoside, madecassic acid and asiatic acid, which impact
on wound healing activity [9]. An investigation into the effects of asiaticoside on human periodontal
ligament cell proliferation, protein synthesis and osteogenic differentiation, revealed periodontal
wound healing activity [10]. Moreover, C. asiatica extract can promote epithelium wound healing in
rabbit corneal epithelial cells [11].
Conventional maceration is the most common approach to extract the compounds with known
biological activities from medicinal plants. There is no prior report of the wound healing activity of the
extract by the supercritical carbon dioxide (scCO2 ) extraction method. Supercritical fluids have been
introduced as an alternative extraction method at low temperature for the preparation of plant extracts.
At the critical point, supercritical fluids have the density of a liquid, but low viscosity with better flow
property like a gas. Carbon dioxide (CO2 ) is a gas widely used to produce supercritical fluid because
of its low critical temperature (Tc = 31.1 ◦ C) and pressure (Pc = 73.8 bar), and provides high solvating
power at the near-critical point [12].
Notably, scCO2 has been used for the substitution of organic solvent in extracting thermal-sensitive
constituents, with the advantages of not only being environmentally friendly, nontoxic and
nonflammable, but also inexpensive [13]. Although the maceration method is simple and low-cost, it
requires a large amount and high purity of the organic solvents, which are usually hazardous and
flammable solvent wastes, and are generally cumbersome [14]. Moreover, extraction by maceration is
nonselective and time-consuming, in comparison to scCO2 extraction.
An important disadvantage of plant extracts in topical products is the plant pigments, which
produce vibrant plant extracts that naturally fade with time, resulting in color variation in the
same product [15]. To overcome this issue, we introduced scCO2 with the cosolvent to extract the
pigments from C. asiatica, and thereby eliminate the dark green appearance of the extract before
ethanolic maceration.
This present study aimed to decrease the presence of plant pigments, and to compare the
composition of the bioactive compounds and their biological activities involved in wound healing
activity (antioxidant screening by 2,2-diphenyl-1-picrylhydrazyl radical [DPPH] and 2,20 -azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid) [ABTS+ ] radical scavenging activities, cell migration assay,
MMP-2 inhibition on human skin fibroblast and endothelial cell tube formation assay) of C. asiatica leaf
extracts obtained by ethanolic maceration with and without pretreatment using scCO2 extraction.
Processes 2020, 8, 277 3 of 15

2. Materials and Methods

2.1. Chemicals
Trolox, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) diammonium salt and L-ascorbic acid standard were obtained from Sigma Chemical Co.
(St. Louis, MO, USA). All other chemical substances were of analytical grade.

2.2. Preparation of Centella asiatica Sample

The leaves of C. asiatica were gathered from Nampu, Ratchaburi, Thailand, during January to
June 2018, by botanists from the Pharmaceutical and Natural Products Research and Development
Unit (PNPRDU), Chiang Mai University, Chiang Mai, Thailand. A herbarium voucher specimen
(PNPRDU004/62) was deposited in the PNPRDU, Faculty of Pharmacy, Chiang Mai University.
The dried leaves were ground into small pieces and kept at 4 ◦ C until use.

2.3. Extraction Method

2.3.1. Conventional Extraction

For conventional extract preparation, the powder of C. asiatica (2 kg) was macerated with 70%
ethanol (1:4.5, w/w) at room temperature for 48 h, following the extraction protocol of Thai National
List of Essential Medicines [16]. Then, the extract was filtered through Whatman paper No. 1, and the
filtrate was evaporated at 50 ◦ C to remove the solvent using a rotary evaporator (Hei-VAP Precision,
Heidolph, Schwabach, Germany) until it was completely dry. The sample was called CV.

2.3.2. Pretreatment Using scCO2

For the pretreatment using scCO2 extraction, the powder of C. asiatica (3.6 kg) was placed into the
supercritical fluid extractor (Guangzhou Gongcheng Digital Science Technology, Guangzhou, China)
at 35 MPa, 60 ◦ C for 3 h, with 95% ethanol (2 kg) as the cosolvent. The volume of the extractor was 24 L.
The ratio of ethanol to CO2 was 1:25 by weight. This process eliminated the color pigments from the
C. asiatica powder. Then, the processed powder of C. asiatica was macerated with 85% ethanol (1:4.5,
w/w) at room temperature for 24 and 48 h to obtain scCO2 extract 1 (SCE1) and 2 (SCE2), respectively.
The 85% ethanol was used in the maceration process, since it gave the highest total triterpene and
triterpenoid glycoside in our preliminary study. Each extract was filtered and evaporated to dryness,
as described in Section 2.3.1.

2.4. Identification of the Active Compounds by High-Performance Thin-Layer Chromatography (HPTLC)

2.4.1. Preparation of Crude Extract and Standard Solution

The C. asiatica extracts (CV, SCE1 and SCE2) were dispersed in 5 mL methanol at 160 µL/mL.
Each sample was sonicated for 10 min and centrifuged to collect the supernatant. The asiaticoside
United States Pharmacopoeia reference standard (USP RS) was dissolved in methanol at 0.5 mg/mL
for standard solution A. The USP RS of powdered C. asiatica extract was dispersed in methanol at
10 mg/mL, sonicated for 10 min and centrifuged. The supernatant was collected and used as standard
solution B.

2.4.2. HPTLC
A Camag high-performance thin-layer chromatography (HPTLC) system (Muttenz, Switzerland)
equipped with an automatic thin-layer chromatography (TLC) sampler ATS 4, thin-layer
chromatography (TLC) scanner 3 and integrated software winCATS version 1.2.3 was used for
the analysis. HPTLC was performed on an HPTLC plate (20 cm × 10 cm) precoated with silica gel
60F254 (layer thickness 0.20 mm).
Processes 2020, 8, 277 4 of 15

The samples (6 µL) and standards (10 µL) were automatically dispensed (TLC sampler ATS 4) on
the plate under a flow of N2 gas, as 10-mm wide bands, positioned 10 mm from the bottom and 10
mm from the side, at an inter-band distance of 35 mm. Linear ascending development was carried
out at room temperature (25 ± 2 ◦ C) and 50 ± 5% relative humidity for 30 min in a Camag twin
trough chamber (20 cm × 10 cm) previously saturated with 10 mL of dichloromethane:methanol:water
(14:6:1, v/v/v). The migration distance was 7.5 cm. The developed TLC plates were air-dried using
an air dryer in a wooden chamber with adequate ventilation. The air-flow in the laboratory was
maintained unidirectional (laminar flow, towards exhaust). The spray reagent was 10% (v/v) sulfuric
acid in methanol. The plate was then oven-heated at 120 ◦ C for 4 min and observed under white
light. The Camag TLC scanner with D2&W lamp at the plate scanning wavelength of 500 nm with
the absorption measurement mode and winCATS software was used for imaging and archiving the
thin-layer chromatograms at the following settings: detector mode, automatic; slit dimensions, 4.0
mm × 0.3 mm; data resolution, 100 µm/step; scanning speed, 20 mm/s. Each sample was analyzed
in triplicate.

2.5. Determination of the Active Compounds by High-Performance Liquid Chromatography (HPLC)

2.5.1. Preparation of Crude Extract and Standard Solutions

According to USP 40-NF 35, the asiaticoside USP RS was dissolved in methanol at 0.05 mg/mL
(standard solution A). The USP RS of powdered C. asiatica extract was dispersed and sonicated in
methanol at 5.0 mg/mL (standard solution B). The 1 g of C. asiatica extracts (CV and SCE1) and methanol
(100 mL) was transferred to a Soxhlet apparatus (GLASSCO, Ambala, India), extracted for 8 h, and
diluted with methanol to 100 mL (sample stock solution). The sample solution was prepared by
diluting sample stock solution (5 mL) with methanol to 10 mL. Before injection, the solutions were
filtered (0.45 µm pore size), discarding the first few milliliters of the filtrate.

2.5.2. HPLC
The total triterpenoids and total triterpenoid glycosides in the standard and C. asiatica extracts
were determined by high performance liquid chromatography (HPLC) using a Luna® C18 column (250
mm × 4.60 mm, 5 µm; Phenomenex, Torrance, CA, USA), LC1200UV/VIS detector and LC1100HPLC
pump. The chromatographic separation was achieved using a binary mobile phase system of 0.3%
(v/v) phosphoric acid and acetonitrile under gradient elution, according to the composition of the
triterpene derivatives of C. asiatica in the USP 40-NF35 monograph. The injection volume and flow rate
were, respectively, 10 µL and 1 mL/min, and the UV detector was set at 200 nm. The content of total
triterpenoids (madecassoside, asiaticoside B, asiaticoside, madecassic acid, terminolic acid and asiatic
acid) and total triterpenoid glucosides (madecassoside, asiaticoside B, asiaticoside) were calculated by
Equation (1):
ru V
Result = × Cs × × D × F × 100 (1)
rs W
ru peak areas of the triterpene derivatives in the sample solution
rs peak areas of asiaticoside in the standard solution A
Cs concentration of USP RS asiaticoside in the standard solution A (mg/mL)
V final volume of sample stock solution (mL)
W weight of C. asiatica used to prepare the sample stock solution (mg)
D dilution factor in preparing the sample solution from the sample stock solution
F conversion factors for analytes: 1.00 for asiaticoside, 1.017 for the sum of madecassoside and
asiaticoside B, 0.526 for the sum of madecassic acid and terminolic acid, and 0.509 for asiatic acid.
Processes 2020, 8, 277 5 of 15

2.6. Determination of Biological Activities of Centella asiatica Extracts

2.6.1. Antioxidant Screening by DPPH Radical Scavenging Activity

The DPPH radical scavenging activity of CV and SCE1 was determined using a previously
described method [17] with slight modifications. Briefly, five serially-diluted concentrations of each
sample, along with 50 µL of distilled water and 50 µL of 0.1 mM ethanolic DPPH solution, were
added to the wells of a 96-well plate (Nalge Nunc International, Rochester, NY, USA). The reaction
mixture was incubated at 25 ± 2 ◦ C for 30 min, then the solution was separated, transferred to a 96-well
microplate, and the absorbance was measured at 515 nm against a blank (ethanol) using a plate reader
(EZ Read 400 Flexi, Biochrom, Cambridge, UK). Ascorbic acid standard was the positive control. All
experiments were performed in triplicate. The percentages of the DPPH radical scavenging activity
were calculated by Equation (2):
 
Acontrol − Asample
DPPH scavenging activity (%) = × 100 (2)
Acontrol

Here Asample is the absorbance of the DPPH radical with the extract, and Acontrol is the absorbance
of the DPPH radical alone. The DPPH scavenging activity (%) of five serially-diluted concentrations
were determined.

2.6.2. Antioxidant Screening by ABTS+ Scavenging Activity

For the ABTS+ assay, the method was used with some modifications [18]. The stock solutions
included ABTS+ solution and potassium persulfate solution at the 1:0.5 molar ratio. To prepare the
working solution, these two stock solutions were mixed in equal quantities and reacted at room
temperature in the dark for 12 h. The solution was then diluted by mixing 1 mL of ABTS+ solution with
distilled water to obtain a spectrophotometric absorbance of 0.7–0.9 at 734 nm. Fresh ABTS+ solution
was prepared for each assay. The extracts (150 µL) were reacted with 2850 µL of ABTS+ solution for
2 h in the dark, and the absorbance was recorded at 734 nm using a 96-well plate reader (EZ Read
400 Flexi, Biochrom, Cambridge, UK). The standard curve was linear between 0.001–1 mg/mL Trolox.
Results are expressed in milligrams of Trolox equivalent antioxidant capacity (TEAC) per gram of
extract. Additional dilution was needed if the ABTS+ value measured was outside the linear range of
the standard curve.

2.7. Wound Healing Activity Assay

2.7.1. Cell Culture

The normal human telomerase reverse transcriptase (hTERT) human fibroblasts (OUMS-36T-2)
and immortalized human umbilical vein endothelial cells (HUEhT-2) were provided by the JCRB
Cell Bank (Osaka, Japan). The normal hTERT human fibroblasts were cultured in complete culture
medium containing Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v)
fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL). HUEhT-2 cells were
cultured in incomplete MCDB 131 medium supplemented with 10% (v/v) FBS, glutamine, endothelial
cell growth supplement (ECGS) and heparin, according to the manufacturer’s instructions. All cells
were incubated in a temperature-controlled and humidified incubator (CCL-050B-8, Esco® , Singapore)
with 5% CO2 at 37 ◦ C.

2.7.2. Determination of Non-Cytotoxic Concentration by the Sulforhodamine B (SRB) Assay

The samples were tested for non-cytotoxic concentration on normal hTERT human fibroblasts and
HUEhT-2 by the SRB assay, as previously described [19]. L-Ascorbic acid (0.00001–0.1 mg/mL) was
Processes 2020, 8, 277 6 of 15

used as a positive control for normal hTERT human fibroblasts. The cells were plated at a density of 1.0
× 105 cells/well in 96-well plates, and left overnight for cell attachment in 5% CO2 atmosphere at 37 ◦ C.
Cells were then exposed to ten serial concentrations of the extracts (in the range of
0.00001–0.1 mg/mL for hTERT human fibroblasts and 0.0005–0.5 mg/mL for HUEhT-2) for 24 h.
After incubation, the adherent cells were fixed in situ, washed, and dyed with SRB. The bound dye was
solubilized, and the absorbance was measured at 540 nm using a 96-well plate reader (EZ Read 400
Flexi, Biochrom). The experiments were conducted in triplicate. The percentages of cell viability were
calculated by Equation (3), where OD denotes optical density:
 
ODsample − ODblank
Cell viability (%) = × 100 (3)
(ODcontrol − ODblank )

2.7.3. Gelatinolytic Activity (Zymography) of MMP-2 Inhibition on Human Skin Fibroblast

The samples (CV and SCE1) were investigated for the gelatinolytic activity of MMP-2 inhibition
in comparison to L-ascorbic acid as the standard. A monolayer of 5 × 105 cells of normal human skin
fibroblasts was maintained in the culture medium without FBS for 24 h, treated with the samples and
L-ascorbic acid standard, respectively, at 0.1 mg/mL, and incubated for 48 h. The culture supernatants
were collected. To assess the gelatinolytic activities of MMP-2 in the culture media, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography was performed using gelatin
as a substrate. Briefly, 20 µL of the cell culture supernatant was suspended in the loading buffer
(0.125 M Tris pH 6.8, 4% SDS and 0.04% bromophenol blue), without prior denaturation and run on
10% SDS polyacrylamide gel containing 1 mg/mL gelatin. After electrophoresis, gels were washed to
remove SDS and incubated for 20 min in the renaturing buffer (50 mM Tris, 5 mM CaCl2 , 0.02% NaN3 ,
2.5% Triton X-100), followed by incubation in developing buffer (50 mM Tris pH 7.5, 5 mM CaCl2 ,
0.02% NaN3 and 1% Triton X-100) at 37 ◦ C for 24 h to allow gelatin degradation by MMPs. Gels were
subsequently stained with 0.5% Coomassie brilliant blue G-250 and de-stained in 30% methanol and
10% acetic acid (v/v) to detect gelatinolytic activity [20]. Gelatinase activity was detected as a white
band on a blue background. The gel was scanned using a gel documentation system (Gel DocTM EZ
Gel, Bio-Rad Laboratories, Hercules, CA, USA) and analyzed by Image LabTM software 5.1 (Bio-Rad
Laboratories, USA). The volume (intensity) multiplied by the number of pixels of the bands on the
gel was determined as the relative MMP-2 content (intensity unit) [21]. The percentages of MMP-2
inhibition relative to the control (the untreated systems) were calculated by Equation (4):

MMP − 2 contentsample
! !
MMP − 2 inhibition (%) = 100− × 100 (4)
MMP − 2 contentcontrol

The assays were done in three independent experiments. The potency of MMP-2 inhibition of the
samples was compared with the positive control (L-ascorbic acid).

2.7.4. Cell Migration Assay

The normal hTERT human fibroblasts were plated in 24-well plates at 1 × 106 cells/well and left
overnight for cell attachment in 5% CO2 atmosphere at 37 ◦ C, as described previously [22,23]. A sterile,
10-µL plastic pipette tip was used to create a linear scratch on the cell monolayer. The cells were washed
once with phosphate-buffered saline (pH 7.4) to remove the cell debris and replaced with the sample in
the incomplete medium. The cell migration was photographed under an inverted microscope at 0 and
12 h. The cell migration area was determined using ImageJ software version 1.52a (NIH, Bethesda,
Processes 2020, 8, 277 7 of 15

MD, USA). The percentage of wound closure and the rate of cell migration were calculated according
to Equations (5) and (6), respectively.
 
Areapre−migration − Areamigration
Wound closure (%) = × 100 (5)
Areapre−migration
 
Areapre−migration − Areamigration
Rate of cell migration = × 100 (6)
Time (h)

2.7.5. Endothelial Cell Tube Formation Assay

An ice-cold 24-well plate was coated with 150 µL/well of Matrigel® Matrix (Discovery Labware, Inc.
Bedford, MA, USA), which was allowed to solidify at 37 ◦ C for 60 min, according to the manufacturer’s
instructions and a previous study [24]. Then, 2 × 105 cells/well of HUEhT-2 were seeded into 24-well
plates, as previously described [24,25]. Incomplete MCDB 131 was used as a culture medium and
negative control. Samples were treated in triplicate in each well, and compared with the positive
control (incomplete MCDB 131 supplemented with glutamine, ECGS, heparin and FBS, according
to the manufacturer’s instructions and a previous study [24]). After incubation at 37 ◦ C and 5%
CO2 for 24 h, tube formation was examined under an inverted phase-contrast microscope. Three
different phase-contrast photomicrographs (10× objective) per well were taken to quantify the length
of capillaries by ImageJ software. The enclosed capillary networks of tube formation were expressed
as a fold increase relative to the cells cultured in the negative control.

2.8. Statistical Analysis

The results were presented as the mean of three independent experiments and analyzed by SPSS
version 17.0. Analysis of Variance (ANOVA) was used for the analysis of the test results (LSD test) at p
< 0.05. The bivariant correlation coefficient (r) was calculated to determine the relationship between
the variables.

3. Results and Discussion

3.1. Physical Characteristic of Centella asiatica Extracts

The C. asiatica extracts (CV, SCE1, SCE2) were green–brown (CV) and yellow–brown (SCE1 and
SCE2) liquids with the density of 1.156, 1.036 and 1.033 g/mL, respectively. The extraction yield of CV
was 34.15% (dry weight basis), while those of SCE1 and SCE2 were 41.67% and 42.87% (dry weight
basis), respectively. The increased yield obtained by 24 and 48 h pretreatment using scCO2 extraction
is possibly because CO2 is a nonpolar molecule, resulting in the enhanced solubility of nonpolar
compounds [26], and the use of ethanol as a cosolvent also allowed to extract polar components [27].
Furthermore, organic solvents that are used at high pressures could enhance the interaction between
the solvent and the matrix, as well as increase the solvent power. Consequently, the mass transfer of
the solutes to the solvent was increased, and in turn, the extraction yields of C. asiatica extracts were
improved [26–28]. Notably, the pretreatment scCO2 extraction with ethanol as a cosolvent destabilized
the dark pigments from the extract, resulting in the yellow–brown SCE extracts [15,29]. A previous
study reported that the ethanol addition to scCO2 promoted certainly the reduction in a- and b-value
in CIE L*, a* and b* (CIE-LAB) values. The reduction of b-value resulted in less yellow product in the
extracts of Pisum sativum L. [30]. The color pigments (e.g., lutein and ß-carotene) might be removed by
ethanol. Due to ethanol as a cosolvent, this might remove polar compounds and cause changes for the
protein structure in plants to release more pigments. Ethanol might promote the affinity between CO2
and pigment molecules by the association pigment–ethanol, decreasing the polarity and increasing the
local density near solute molecules [31].
Processes 2020, 8, 277 8 of 15

The scCO2 is a green technology. However, it is not perfectly suitable for the extraction of polar
organic compounds
Processes 2020, 8, 277 including alcohols and/or high molecular components, such as8 saponins, of 16 phenolic
compounds and terpenoids from plants [32]. The scCO2 and ethanol as a cosolvent used in the
3.2. Identification of the Bioactive Compounds by HPTLC
pretreatment allowed us to extract both moderately polar and nonpolar compounds, combined with
The composition
short extraction of the
times and biologically-active
high penetrating of compounds was in
the solvent compared among the
the extraction C. asiatica
[33], which compared to
extracts (CV, SCE1 and SCE2) by using HPTLC. Figure 1A shows the chromatogram of the USP RS
conventional extraction for producing a variety of natural product extracts [30].
asiaticoside (track 1), SCE1 (track 2), SCE2 (track 3), CV (track 4) and the USP RS powdered C. asiatica
extract (track 5). Asiaticoside was the major component in all extracts, but presented in different
3.2. Identification
amounts. It had of the
thesame
Bioactive Compounds
retention bythe
factor (Rf) in HPTLC
TLC fingerprint of each extract (see Figure 1B),
and no unique peaks occurred in SCE1 and SCE2 when compared with CV.
The composition of the biologically-active compounds was compared among the C. asiatica extracts
In this study, C. asiatica was pretreated using scCO2 extraction with ethanol as a cosolvent,
(CV, SCE1 andbySCE2)
followed ethanolicby maceration
using HPTLC. for 24 Figure
and 48 h1A showsSCE1
to obtain the chromatogram of theSeveral
and SCE2, respectively. USP RS asiaticoside
(trackreports
1), SCE1 (track
showed that2),
theSCE2 (track
ethanolic 3), CVby(track
extraction 4) and
maceration the USP
produced theRS powdered
same C. asiatica extract (track
type of biologically-
active compounds,
5). Asiaticoside was the but major
in different amounts relative
component to the pretreatment
in all extracts, using scCO
but presented 2 extraction [34–
in different amounts. It had the
37]. Here, SCE1 was selected for further analysis because of its greater yield than SCE2, and the same
same retention factor (Rf) in the TLC fingerprint of each extract (see Figure 1B), and no unique peaks
profile of compounds between SCE1 and SCE2 was found in the HPTLC chromatogram.
occurred in SCE1 and SCE2 when compared with CV.

(A)

(B)

USP Asiaticoside RS SCE1 SCE2 CV USP powdered C. asiatica extract

Figure 1. (A) High-Performance Thin-Layer Chromatography (HPTLC) chromatogram and (B) HPTLC
fingerprint of Centella asiatica extracts, USP Asiaticoside RS (track 1), C. asiatica extracts from the
pretreatment of scCO2 method, SCE1 (track 2), SCE2 (track 3), C. asiatica extracts from conventional, CV
(track 4) and the USP powdered C. asiatica extract (track 5).
Processes 2020, 8, 277 9 of 15

In this study, C. asiatica was pretreated using scCO2 extraction with ethanol as a cosolvent, followed
by ethanolic maceration for 24 and 48 h to obtain SCE1 and SCE2, respectively. Several reports showed
that the ethanolic extraction by maceration produced the same type of biologically-active compounds,
but in different amounts relative to the pretreatment using scCO2 extraction [34–37]. Here, SCE1 was
selected for further analysis because of its greater yield than SCE2, and the same profile of compounds
between SCE1 and SCE2 was found in the HPTLC chromatogram.

3.3. HPLC Profile of Bioactive Compounds

The compounds with known biological activities in the C. asiatica extracts (CV and SCE1) are
shown in Table 1. The contents of madecassoside, asiaticoside, madecassic acid, terminolic acid and
asiatic acid in SCE1 were different from those in CV, resulting in noticeably higher total triterpenoid
glycosides and total triterpenes when the samples were pretreated using scCO2 (p < 0.05). This enriched
amount of active compounds from C. asiatica extracted with scCO2 might be explained by the increased
contact surface associated with the degradation of the plant cell wall during comminution of the
sample, which promoted the solvent–solute interactions during scCO2 extraction [26]. Besides the
increased content of total triterpenes in the C. asiatica extracts, extraction with scCO2 eliminated the
pigments. A previous study reported that the approximate 30 MPa pressure of scCO2 was incapable of
extracting the pigments from the seed tissues. High pressure of approximately 50 MPa, and the use
of a low polar modifier, was necessary to dissociate chlorophylls from marine algae [38,39]. At the
pressure of 35 MPa used in the current study, the decreased solvent power and increased diffusivity of
CO2 lowered the content of pigments in the extracts.

Table 1. Biological compound contents of Centella asiatica extracts from conventional (CV) and the
pretreatment of scCO2 method (SCE1).

Biological Compound C. asiatica Extract C. asiatica Extract

(% w/w) (Conventional; CV) (scCO2 ; SCE1)
Madecassoside 0.696 ± 0.004 a 0.655 ± 0.002 a
Asiaticoside B 0.964 ± 0.007 0.964 ± 0.005
Asiaticoside 1.144 ± 0.007 b 1.179 ± 0.005 b
Total triterpenoid glycoside 2.795 ± 0.016 2.799 ± 0.011
Madecassic acid 0.135 ± 0.001 c 0.426 ± 0.006 c
Terminolic acid 0.094 ± 0.004 d 0.307 ± 0.003 d
Asiatic acid 0.203 ± 0.006 e 0.576 ± 0.003 e
Total triterpene 3.228 ± 0.012 f 4.107 ± 0.018 f
Note: a–f mean significant different at p-value < 0.05.

3.4. Biological Activity of Centella asiatica Extracts

Table 2 shows the biological activities (DPPH and ABTS+ radical scavenging activities) were
higher for CV (46.85 ± 2.31% and 0.017 ± 0.004 mg TEAC/g) than SCE1, which might be due to the
high solubilizing power of scCO2 , which was applied 3 h prior to the ethanolic maceration process
in the C. asiatica extraction (SCE1). The Trolox standard curve in the ABTS+ scavenging assay was
y = −7.547x + 1.4813, r2 = 0.995.
Moreover, the total phenolic content was greater for CV (1.391 ± 0.044 µg gallic acid equivalents
[GAE]/g extract) than SCE1 (0.953 ± 0.031 µg GAE/g extract). The scCO2 extraction method with ethanol
as a cosolvent could have facilitated dissolution of the polar compounds, such as those containing
phenolic groups, in the SCE1 extract, leading to the relatively lower total phenolic content, and
antioxidant activity (DPPH and ABTS+ ). The trisaccharides, namely asiaticoside and madecassoside,
have higher polarities than the aglycones, which include madecassic acid and asiatic acid, leading to
the lower abundance of these compounds in SCE1 than CV [40].
 Processes 2020, 8, 277 10 of 15

Table 2. Biological activities of Centella asiatica extracts from conventional and scCO2 method (SCE1).

DPPH Scavenging Activity ABTS Scavenging Activity

Biological Activity
(%) (mg TEAC/g)
C. asiatica extract (conventional; CV) 46.85 ± 2.31 0.017 ± 0.004
C. asiatica extract (scCO2 ; SCE1) 37.52 ± 1.35 0.010 ± 0.002
standard L-ascorbic acid 95.41 ± 1.39 N/A
Note: the concentration of C. asiatica extracts from conventional and scCO2 method were 1 mg/mL, N/A: not available.

3.5. Wound Healing Activity

The non-cytotoxic concentration of C. asiatica extracts (CV and SCE1), as assessed by the SRB assay,
was 0.01 mg/mL in both the normal hTERT human fibroblasts and HUEhT-2 cell line. This concentration,
which provided more than 90% cell viability, was selected for further wound healing analyses.

3.5.1. MMP-2 Inhibition Activity

The inhibitory effects of the gelatinolytic activity on MMP-2 expression found for the C. asiatica
extracts (CV and SCE1) and the ascorbic acid standard, are illustrated in Figure 2. The faded band of
MMP-2 inhibition was not due to cell cytotoxicity, because all samples were non-cytotoxic to human
skin fibroblasts by the SRB assay. In comparison to the control, SCE1 exhibited the highest MMP-2
inhibition of 58.48 ± 7.50%, followed by CV (50.74 ± 7.31%) and then the ascorbic acid standard (31.20
± 2.18%).
Processes 2020, 8, 277 11 of 16

(A)

(B)
% MMP-2 inhibition
70
% MMP-2 inhibition activity

60
50
40
30
20
10
0
Standard ascorbic SCE CV Negative control
-10 acid

Figure 2. Matrix metallopeptidases 2 (MMP-2) inhibition activity of Centella asiatica extracts from the
Figure 2. Matrix
pretreatment ofmetallopeptidases (MMP-2) inhibition
scCO2 (SCE1) and2 conventional method activity of Centella to
(CV) compared asiatica extracts
standard from acid.
ascorbic the
pretreatment of scCO 2 (SCE1) and conventional method (CV) compared to standard ascorbic acid.
(A) Zymograms and (B) the percentages of MMP-2 inhibition.
(A) Zymograms and (B) the percentages of MMP-2 inhibition.
MMPs may also have negative effects on cell proliferation in the wound healing process [9].
3.5.2. Cell Migration
The extracts and Angiogenesis
that presented wound healing Activity
activity should show high inhibition of MMP-2 expression.
As mentioned
Table 3 showsabovethe(Section 1), the normal
cell migration wound healing
and angiogenesis ofisC.a complex processrelative
asiatica extracts involvingtoinflammation,
the positive
and negative controls. Consistent with the trend of MMP-2 inhibition, SCE1 induced extracellular
proliferation and remodeling/maturation phases. During the maturation of the wound, the higher cell
matrix undergoes
migration (59.83 ± certain
1.85% of changes,
initial) followed by scar formation,
and the number which ±marks
of vessels (18.25 4.58) intheangiogenesis
physiologicalactivities
endpoint
of healing.with
compared TheCVendpoint
(50.43 is also directly
± 6.22% proportional
of initial and 16.50 to the extent
± 3.51, of inflammatory
respectively). processesmight
This observation occurring
be
throughout the healing. The MMPs, which have an important role in tissue
due to the higher content of asiaticoside, total triterpenoid glycosides, madecassic acid, terminolicremodeling, eliminate
damaged
acid, asiaticprotein,
acid and destroy the provisional
total triterpenes in SCE1extracellular matrix,
than CV. These facilitate
active its migration
compounds to the center
in C. asiatica play anof
the wound,
important remodel
role the granulation
in the wound tissue, regulate
healing process, thein
especially activity of some [41,43,44]
cell migration growth factors and probably
and angiogenesis
[7,45]. Moreover, the synergistic effect of these active compounds in C. asiatica could be tissue
control angiogenesis [3]. In earlier research, MMP-2 and MMP-9 decreased in the wound observedtreated
in
the wound healing activity. Lu et al. (2004) reported that asiaticoside could stimulate the mRNA
levels and increase the protein production of certain genes responsible for extracellular matrix
synthesis encoding type I and type III collagen proteins in human fibroblasts. Furthermore,
asiaticoside may control the cell-cycle progression and cell proliferation that enhance cell migration
and angiogenesis activity [46].
Processes 2020, 8, 277 11 of 15

with the triterpenoid saponin-rich fraction of C. asiatica. MMP-2 and MMP-9 regulate the turnover
of the matrix proteins by degrading basement membrane proteins, which include collagen IV and
V, gelatin, elastin and fibronectin [41]. Other work demonstrated that asiatic acid and triterpene
compounds reduced the UVA-induced activation and expression of MMP-2, using HaCaT human
keratinocytes as a model cell system [42].

3.5.2. Cell Migration and Angiogenesis Activity

Table 3 shows the cell migration and angiogenesis of C. asiatica extracts relative to the positive and
negative controls. Consistent with the trend of MMP-2 inhibition, SCE1 induced higher cell migration
(59.83 ± 1.85% of initial) and the number of vessels (18.25 ± 4.58) in angiogenesis activities compared
with CV (50.43 ± 6.22% of initial and 16.50 ± 3.51, respectively). This observation might be due to the
higher content of asiaticoside, total triterpenoid glycosides, madecassic acid, terminolic acid, asiatic
acid and total triterpenes in SCE1 than CV. These active compounds in C. asiatica play an important role
in the wound healing process, especially in cell migration [41,43,44] and angiogenesis [7,45]. Moreover,
the synergistic effect of these active compounds in C. asiatica could be observed in the wound healing
activity. Lu et al. (2004) reported that asiaticoside could stimulate the mRNA levels and increase the
protein production of certain genes responsible for extracellular matrix synthesis encoding type I and
type III collagen proteins in human fibroblasts. Furthermore, asiaticoside may control the cell-cycle
progression and cell proliferation that enhance cell migration and angiogenesis activity [46].

3.6. Relationship between the Biological Activities and Bioactive Compound Contents in Centella
asiatica Extracts
Pearson’s correlation coefficients (r) between the biological activities (the cell migration, number
of vessels and angiogenesis) and the content of bioactive compounds in the C. asiatica extracts (SCE1)
are presented in Table 4. The correlations based on Akoglu’s (2018) classification of the correlation
coefficient as particularly high (r > 0.9), high (0.9 > r > 0.7), moderate (0.7 > r > 0.5) and poor (r <
0.5) [47]. There was a strong and significant linear relationship between the DPPH radical scavenging
activity and the content of madecassoside (r = 0.914, p < 0.01). This significant positive correlation
implied that the antioxidant activity of SCE1 varied proportionally to change of concentration of
madecassoside. Numerous studies also reported that madecassoside from other plant has been shown
to exert various pharmacological activities, including antioxidative properties [48,49]. Asiaticoside
and cell migration were strongly correlated (r = 0.854, p < 0.05). This result agreed with a previous
study, showing that asiaticoside enhanced the migration rate of normal human dermal fibroblasts.
Furthermore, the initial skin cell adhesion and cell proliferation were also increased by asiaticoside [50].
Therefore, the yield of madecassoside and asiaticoside can be used to predict the antioxidant activity of
SCE1 due to their good correlation with antioxidant activity, whereas asiaticoside might be attributed
to predict the cell migration potential.
 Processes 2020, Processes
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277 8, 277
2020, 8, 277 12 of 17
12 of 1712 of 17

Processes 2020, Processes

Processes8,2020,
277 8, 277
2020, 8, 277 12 of 17
12 of 1712 of 17

Processes
Processes 2020,
Processes
2020, 8,2020,
8, 277 277 8, 277
Processes 2020, 8, 277
TableTable
3. Cell3.Table
migration and
3. Cell angiogenesis
migration
Cell migration of Centella
and angiogenesis
and angiogenesis asiatica extracts
of Centella
of Centella asiatica fromextracts
asiatica
extracts conventional and scCO
from conventional
from conventional method
and
and 2scCO (SCE1).
scCO 2 method
2 method (SCE1).(SCE1). 12 of 17
12 of 1712 12
of of
1715

TableTable
3. Cell3.Table
migration and
Cell migration
3. Cell angiogenesis of Centella
and angiogenesis
migration of Centella
and angiogenesisasiatica extracts
asiatica
of Centella from
extracts
Cell asiatica
Migration
Cell conventional
fromMigration
Cell
Migration and scCO
conventional
extracts method
and 2scCO
from conventional and (SCE1).
2 method
scCO (SCE1).(SCE1).
2 method
Sample
SampleSample At Initial At Initial AfterAfter
At Initial 24 h 24 After
h 24 h Angiogenesis Activity
Angiogenesis
Angiogenesis Number
Activity
Activity of Number
Number Vessels of Vessels
of Vessels
TableTable
3. Cell migration
Cell3.Table and
3. Cell
Cell migration
migration angiogenesis
migration
and and of Centella
and angiogenesis
angiogenesis
angiogenesis ofof asiatica
Centella
Centella (% of
extracts
of asiatica
Centella
asiatica
Cell Initial)
(%
fromof
asiatica
extracts
Migration
Cell (%
fromof
Initial)
Migration
extracts Cell
from Initial)
conventional
extracts and scCO
from conventional
conventional
Migration
conventionaland
andmethod
and
2scCO (SCE1).
scCO 2 method
2 method
scCO (SCE1).(SCE1).
Table 3. 2 method (SCE1).
Sample
SampleSample At Initial At Initial AfterAfter
At Initial 24 h 24 h 24 h
After Angiogenesis Activity
Angiogenesis Number
Activity
Angiogenesis of Number
Number
Activity Vessels
of Vessels
of Vessels
(% of
Cell Initial)
(% of
Migration
Cell Initial)
(% of
Cell Initial)
Migration
Migration
Sample
SampleSample At Initial At Initial AfterAfter
At Initial 24 h 24 After
h 24 h Angiogenesis
Cell Migration Activity
Angiogenesis
Angiogenesis Number
Activity
Activity of Number
Number Vessels of Vessels
of Vessels
Sample At Initial After 24 h (% of(% Initial)(% of Initial)
of Initial) Angiogenesis Activity Number of Vessels
(% of Initial)
C. asiatica extract
C. asiatica
C. asiatica extractextract
50.43 50.43
± 6.22±50.43
6.22 ± 6.22 16.50 16.50
± 3.51±16.50
3.51 ± 3.51
(conventional;
C. asiatica CV)
(conventional;
(conventional;
extract
C. asiatica CV)
extract
C. asiatica CV)
extract
50.43 50.43
± 6.22±50.43
6.22 ± 6.22 16.50 16.50
± 3.51±16.50
3.51 ± 3.51
(conventional;
C. asiatica
C. asiatica C. C.CV)
(conventional;
extract
asiatica
extract CV)
(conventional;
asiatica
extract CV)
extract
50.43 50.43
± 6.22±50.43
6.22
50.43 ± 6.22
± 6.22 16.50 16.50
± 3.51±16.50
3.51 ± 3.51
16.50 ± 3.51
(conventional; CV) CV) CV)
(conventional;
(conventional;
(conventional; CV)

C. asiatica extract
C. asiatica
C. asiatica extractextract
59.83 59.83
± 1.85±59.83
1.85 ± 1.85 18.25 18.25
± 4.58±18.25
4.58 ± 4.58
C.(scCO 2; SCE1)
(scCO
asiatica
C.
C. asiatica extract 2(scCO
;
extract
asiatica
C. SCE1) 2; SCE1)
extract
asiatica extract
59.83 59.83
± 1.85±59.83
1.85
59.83 ± 1.85
± 1.85 18.25 18.25
± 4.58±18.25
4.58 ± 4.58
18.25 ± 4.58
(scCO (scCO
; ;
SCE1)
2
C. asiatica SCE1)
(scCO 2;
extractSCE1)
(scCO
C. asiatica
2 C. asiatica 2; SCE1)
extractextract
59.83 59.83
± 1.85±59.83
1.85 ± 1.85 18.25 18.25
± 4.58±18.25
4.58 ± 4.58
(scCO(scCO
2; SCE1)
2(scCO 2; SCE1)
; SCE1)

Positive
Positive control
control Positive
Positive controlcontrol 58.23 58.23
± 10.50
±58.23
10.50
58.23 ± 10.50
± 10.50 45.75 45.75
± 6.03±45.75
6.03 ± 6.03
45.75 ± 6.03
Positive control
Positive controlcontrol
Positive 58.23 58.23
± 10.50
±58.23
10.50± 10.50 45.75 45.75
± 6.03±45.75
6.03 ± 6.03
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277 8, 277
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13 of 1713 of 17
Positive control
Positive
Positive controlcontrol 58.23 58.23
± 10.50
±58.23
10.50± 10.50 45.75 45.75
± 6.03±45.75
6.03 ± 6.03

Negative
Negative control
Negative controlcontrol
Negative
control 25.87 25.87
± 10.41
±25.87
10.41
25.87 ± 10.41
± 10.41 0 0 0 0

Note: positive control for cell migration assay was L-ascorbic acid, for angiogenesis was VEGF.
Note: Note:
positive control
positive
Note: for cell
control
positive migration
for cell for
control assay
migration
cell was L-ascorbic
assay
migrationwas acid,
L-ascorbic
assay was for angiogenesis
acid,
L-ascorbic foracid, was VEGF.
angiogenesis was VEGF.
for angiogenesis was VEGF.
Processes 2020, 8, 277 13 of 15

Table 4. Pearson’s correlation coefficients between the biological activities and the content of bioactive
compounds in Centella asiatica extracts (SCE1).

DPPH ABTS MMPI MG AG

DPPH 1
ABTS 0.662 1
MMPI −0.343 −0.358 1
MG −0.799 −0.369 0.701 1
AG −0.229 0.298 0.362 0.392 1
M 0.914 * 0.803 −0.610 −0.780 −0.198
AB −0.232 0.398 −0.168 0.415 0.044
A −0.980 ** −0.621 0.461 0.854 * 0.276
MA −0.950 ** −0.808 0.533 0.781 0.163
TA −0.949 ** −0.807 0.526 0.769 0.185
AA −0.948 ** −0.798 0.545 0.788 0.181
TG −0.448 −0.013 −0.630 0.068 0.039
T −0.956 ** −0.801 0.517 0.779 0.173
Note: * and ** indicate the significant level at 0.05 and 0.01 level, respectively. MMPI: matrix metallopeptidases 2
(MMP-2) inhibition activity; MG: migration; AG: angiogenesis; M: madecassoside; AB: asiaticoside B; A: asiaticoside;
MA: madecassic acid; TA: terminolic acid; AA: asiatic acid; TG: triterpenoid glycoside; T: triterpene.

4. Conclusions
Pretreatment of C. asiatica using scCO2 extraction with ethanol as the cosolvent prior to ethanolic
maceration can eliminate the plant pigments, avoiding color fading and color variation in the products,
and provide an extract with greater in vitro wound healing activity than the conventional method.

Author Contributions: Conceptualization, W.R.; methodology, W.R.; software, C.K.; validation, K.S.; formal
analysis, P.J.; investigation, C.K.; resources, W.R.; data curation, S.S.; writing—original draft preparation, W.R.
and C.K.; writing—review and editing, W.R. and C.K.; supervision, W.R.; project administration, W.R.; funding
acquisition, W.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research was partially supported by Chiang Mai University, Thailand, with a grant number 6/2562.
Acknowledgments: This study was partially supported by Chiang Mai University, Thailand. We are very thankful
to Bangkok Lab and Cosmetic Co., Ltd. for C. asiatica powder.
Conflicts of Interest: The authors declare no conflict of interest.

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