LAPORAN AKHIR PRAKTIKUM

KIMIA BAHAN ALAM I

PEMERIKSAAN SENYAWA-SENYAWA METABOLIT SEKUNDER
( STEROID, FLAVONOID, TERPENOID, SAPONIN DAN FENOLIK )
TERHADAP UVARIA CHAMAE

Disusun Oleh :
ADINDA PUTRI KANA
NIM 21.01.01.186

DOSEN PEMBIMBING: APT. EMA RATNA SARI, M.FARM

SEKOLAH TINGGI ILMU FARMASI

BHAKTI PERTIWI PALEMBANG
2023
 BAB I
PENDAHULUAN

1.1 Latar Belakang

Puncak Anailand merupakan salah satu objek wisata yang berada di wilayah Kabupaten
Padang Pariaman, Sumatera Barat. Lokasi wisata alam Puncak Anai ini mudah diakses
karena berada di jalan trans utama yang menghubungkan Padang - Bukittinggi. Jadi sayang
jika dilewatkan, karena banyak hal menarik jika kita bisa menyempatkan diri berwisata ke
Puncak Anai ini. Puncak Anailand dengan panorama alam yang terletak di puncak bukit
dikandang ampek kenagarian guguak kecamatan kayutanam 2x11 kabupaten padang
pariaman, diketinggian 650 mdpl.
Hutan primer (primary forest) adalah hutan yang telah mencapai umur lanjut dan ciri
strktural tertentu yang sesuai dengan kematangan serta sifat-sifat ekologis yang unik
(Rangkuti, dkk, 2012). Melalui ekspolrasi yang dilakukan bahwa hutan pada kawasan lembah
harau memiliki deversitas yang baik mulai dari tumbuhan tingkat tinggi hingga rendah
seperti tumbuhan dari Pteridophyta.
Eksplorasi fauna di kawasan hutan Harau merupakan bentuk aplikatif dari
pembelajaran teoritis pada Mata Kuliah Kimia Bahan Alam. Kegiatan kuliah lapangan ini
selain megumpulkan dan mengamati spesimen flora hutan, dilakukan pula penyimpanan
spesimen menjadi herbarium dan secara konvensional dilakukan pula pengujian fitokimia
terhadap sampel segar dari tiap tanaman yang dikumpulkan.

1.2 Tujuan Kuliah Lapangan

Adapun kuliah lapangan ini bertujuan untuk mengetahui dan melakukan skrining awal
berupa pengujian kualitratif terhadap potensi-potensi kandungan fitokimia pada flora di
Kawasan Lembah Anai, Sumatera Barat.
1.3 Manfaat Kuliah Lapangan

Mahasiswa memiliki kecakapan dalam melakukan prosedural skrining awal terhadap

sampel yang ditemukan dari kawasan hutan Harau. Baik pada tahap identifikasi sempel,
pembutan herbarium, dan pengujian fitokimia.
 BAB II

TINJAUAN PUSTAKA

Gambar 2.1 Uvaria chamae

2.1 Klasifikasi Tanaman

Kingdom : Plantae
Divisi : Tracheophytes
Kelas : Angiosperms
Ordo : Magnoliales
Famili : Annonaceae
Genus : Uvaria L.
Spesies : Uvaria chamae (wikipedia, 2023)

2.2 Deskripsi

Uvaria chamae, umumnya dikenal sebagai akar jari atau pisang semak termasuk
dalam keluarga Annonaceae. U.chamae adalah tanaman hijau yang tumbuh hingga
ketinggian 3,6-4,5 m. Daunnya menjari, ujung daun menjari 2 dan pertulangan daunnya
gundul. Ini adalah pohon kecil yang berasal dari hutan hujan tropis di Afrika Barat dan
Tengah. Afrika Barat dan Tengah yang tumbuh di semak-semak basah dan pesisir
pantai. Semua bagian tanamannya harum, akar dan kulit akarnya memiliki reputasi
yang tersebar luas. Akarnya (ditumbuk atau ditumbuk) digunakan untuk pengobatan
mimisan, jantung
penyakit (bronkus, paru-paru, dll.), dan darah dalam urin, tumpukan dan demam. Zat-
zat alami ini terdistribusi secara luas dalam spesies tanaman dan diakui melalui
manfaatnya
efek kesehatan yang bermanfaat.

2.3 Nama Umum

Uvaria chamae, umumnya dikenal sebagai akar jari atau pisang semak adalah
semak besar yang merambat atau pohon kecil yang berasal dari Afrika Barat dan
Tengah yang beriklim tropis, yang tumbuh di hutan basah dan kering serta semak
belukar pantai. Nama umum mengacu pada buah yang tumbuh dalam tandan kecilnya;
buahnya dapat dimakan dan dimakan secara luas.

2.4 Kandungan Kimia

Hasil ini mengungkapkan bahwa ekstrak botn kaya akan ini fitokimia. Namun
kandungan fenol total yang tinggi dicatat dalam kaitannya dengan flavonoid dan tanin.
Selain itu ekstrak etanolik memiliki senyawa uji fitokimia konten (9.51 ± 0.96 µg
hingga 21.14 ± 0.51µg) daripada air ekstrak air (6,253±1,10 µg hingga 8,917±0,92
µg.). Hasil penelitian ini berbeda dengan hasil yang diperoleh oleh Donatus et
al,200922. Pada penelitian ini, kandungan total fenol, flavonoid dan tanin dari ekstrak
akar Uvaria chamae berturut-turut adalah 0,10±0,30 µg, 5,70±0,03 µg dan 0,40±0,03
µg. Perbedaan konsentrasi ini bisa jadi dapat dijelaskan oleh beberapa faktor termasuk
metode yang digunakan.
Studi terbaru telah menunjukkan faktor intrinsik yang mungkin menyebabkan
perbedaan ini. Faktor-faktor tersebut adalah faktor geografis, iklim dan faktor genetik
tetapi juga tingkat kematangan tanaman.

2.5 Khasiat Tanaman

Manfaat kesehatan dari tanaman obat dikaitkan dengan sebagian disebabkan oleh
komposisi fitokimia yang unik (Okwu, 2005). Fitokimia adalah metabolit sekunder dari
asal tanaman yang bertindak sebagai antioksidan dan merangsang enzim pelindung di
hati atau memblokir kerusakan pada materi genetik (Okwu, 2004). Mereka mencegah
terjadinya spesies kimia oksidatif, merangsang mekanisme perbaikan antioksidan dan
kapasitas pemulungan radikal bebas dalam sistem.

2.6 Uji Fitokimia

2.6.1 Tujuan Percobaan
Agar mahasiswa mampu memahami dan mampu melakukan sendiri bagaimana
mengidentifikasi dan mendeteksi bermacam-macam tumbuhan dengan uji gitu kimia
atau uji kandungan kimia di lapangan
2.6.2 Tinjauan Pustaka
2.6.2.1 Alkaloid
Alkaloid adalah senyawa yang mengandung substansi dasar nitrogen basa
biasanya dalam bentuk cincin heterosiklik Alkaloid terdistribusi secara luas pada
tanaman diperkirakan sekitar 15 sampai 20% pastilah tanaman mengandung lah kalau
banyak Alkaloid merupakan turunan asam Amino lisin nikotin berani liat asam Amino
di sintetis dalam tanaman dengan proses Dekarboksilasi menjadi Aminah Aminah
kemudian di rubah menjadi Aldehida oleh Amina oksida Alkaloid biasanya pahit dan
sangat beracun. Alkaloid ini diklasifikasikan lagi berdasarkan tipe dasar kimia pada
nitrogen yang terkandung dalam bentuk heteroksiklik Klasifikasi alkaloid tersebut
meliputi irolizidine alkaloids, peperidine alkaloids, pyridine alkaloids, indole alkaloids,
quinolizidine alkaloids, steroid alkaloids, policyclic diterpene alkaloids, tryptamine
alkaloids, tropane alkaloids, fescue alkaloid dan miscellaneous alkaloid Peran alkaloid
dalam jaringan tanaman tidak pasti, mereka telah dikenal sebgai produk metabolik atau
substansi. Alkaloid dapat digolongkan dalam 3 golongan yaitu (Rizal, 2011) :
1. Alkaloid sejati yaitu senyawa yang mempunyai cincin nitrogen heterosiklik, bersifa
basa dan berasal dari asam amino.
2. Alkaloid gabungan yaitu turunan asam amino, atom nitrogennya tidak dalam bentuk
cicin heterosiklik. Alkaloid gabungan bersifat basa, dialam diturunksn dalam
biosintesis asam amino itu sendiri. Contohnya meskaline.
3. Alkaloid semu yaitu basa tumbuhan yang mengandung nitrogen heterosiklik,
memilikii aktifitas dantidak mempunyai hubungan biosintesis dengan asam amino.
Alkaloid semu diturunkan dari senyawa-senyawa terpenoid turunan asam asetat
poliketonlifatik. Contohnya kafein yang terdapat pada kopi.
2.6.2.2 Flavonoid
Flavonoid adalah suatu kelompok yang termasuk ke dalam senyawa fenol yang
terbanyak dialam, senyawa-senyawa flavonoid ini bertanggung jawab terhadap zat
warna ungu, merah, biru, dan sebagian zat warna kuning dalam tumbuhan. Berdasarkan
strukturnya senyawa flavonoid merupakan turunan induk" flavon" yakni nama sejenis
flavonoid yang tersebar jumlahnya dan lizim ditemukan, yang terdapat berupa tepung
putih pada tumbuhan primula. Sebagian besar flavonoid yang trdapat pada tumbuhan
terikat pada molekul gula sebagai glikosida, dan dalam bentuk campuran, jarang sekali
dijumpai berupa senyawa tunggal Disamping itu sering ditemukan campuran yang
terdiri dari flavonoid berbeda kelas (Putri, 2011).
2.6.2.3 Terpenoid
Golongan senyawa ini dapat dipisahkan dari tumbuhan sumbernya melalui
destilaso uap atau secra ekstraksi dan dikenal dengan nama minyak atsiri. Beberapa
contoh minyak atsiri, misalnya minyak yang diperoleh dari cengkeh, bunga mawar,
serai kamfe, sedar (tumbuhan cedrus) dan sirih Senyawa organik bahan alam golongan
minyak atsiri sangat banyak digunakan dalam industri wangi-wangian, makanan dan
obat-obatan Banyak tumbuhan (bunga, daun, biji atau akar) yang berbau harum. Bau
harum itu berasal dari senyawa yang terdiri dari 10 dan 15 karbon yang disebut
terpenoid.
2.6.2.4 Steroid
Senyawa steroid adalah senyawa turunan (derivat) lipid yang tidak terhidrolisis.
Senyawa yang terdapat steroid, misalnya kolesterol, ergosterol, danestrogen. Pada
umumnya steroid berfungsi sebagai hormon. Secara sederhana steroid dapat diartikan
sebagai kelas senyawa organic bahan alam yang kerangka srtukturnya berfungsi sebgai
hormon. Secara sederhana dapat diartikan sebagai kelas senyawa organik bahan alam
yang kerangka strukturnya terdiri dari androstan (klopentanofenantren, mempunyai
empat cincin perdu). a. Mengandung gugus fungsi oksigen (sebagai O atau OH) pada C
b. Mengandung gugus samping pada C17 c. Banyak yang mengandung ikatan rangkap
C.-C, atau C, C.
2.6.2.5 Saponin
Saponin merupakan suatu senyawa glikosida kompleks yaitu senyawa hasil
kondensasi suatu gula dengan suatu senyawa hidroksil organik yang apabila dihidrolisis
akan menghasilkan gula (glikon) dan non gula (aglikon), saponin ini terdiri dua
kelompok saponin triterpenoid dan saponin steroid. Saponin banyak digunakan untuk
bahan pencuci kain (batik) dan sebgai shampo. Saponin dapat diperoleh dari tumbuhan
melalui metode ekstraksi. Senyawa metabolit sekunder yang terdapat dalam tumbuhan
merupakan zat bioaktif yang berkaitan dengan kandungan kimia dalam tumbuhan,
sehingga sebagian dapat digunakan sebagai bahan obat.
2.6.2.6 Fenolik

Senyawa fenolik merupakan senyawa yang banyak ditemukan pada tumbuhan.

Fenolik memiliki cincin aromatik satu atau lebih gugus hidroksi (OH). Senyawa ini
diberi nama berdasarkan nama senyawa induknya, fenol. Senyawa fenol kebanyakan
lebih dari satu sehingga disebut polifenol. Senyawa fenolik meliputi aneka ragam
senyawa yang berasal dari tumbuhan yang mempunyai ciri sama, yaitu cincin aromatik
yang mengandung satu atau dua gugus OH.

2.7 Prosedur Kerja

2.7.1 Alat dan Bahan yang digunakan:
1. Alat-alat :

- Pipet tetes besar - Bunsen

- Tabung reaksi besar - Penjepit kayu
- Pipet tetes kecil - Korek api
- Tabung reaksi besar - Botol reagen
- Lumpang dan alu - Label
- Plat tetes - Spatel
- Kapas - Pot salep
- Gunting - Rak tabung.
2. Bahan-bahan:

- Asam Sulfat - Norit

- Sampel - Reagen Mayer
- Asam Sulfat pekat - Logam Mg
- Kloroform - Aquadest
- Etanol - HCI
- FeCl3 - Pasir Bersih
- Kloroform Amoniak - Asam Asetat Anhidrat

2.7.3 Cara Kerja Fitokimia

1. Alkaloid
a) Potong kecil-kecil 2-4 daun segar yang telah disiapkan dan disimpan dalam kanton
plastik Kemudian dihaluskan dalam lumpang dengan menambahkan secukupnya pasir
bersih dan 10 ml kloroform.
b) Setelah di gerus halus kemudian tambahkan 10 ml kloroform amoniak 0,005 N
diaduk/digerus perlahan.
c) Saring larutan dengan kapas sebgai penyaring, sedot larutan dengan menggunkan
pipet tetes, masukkan ke daam tabung reaksi.
d) Tambahakan 10 tetes asam sulfat dan balik-baikan tabung perlahan (kocok perlahan)
biarkan sejenak hingga terjadi pemisahan lapisan asam dan lapisan kloroform
2. Flavonoid
a) Ambil kira-kira sebgaian dari lapisan air (1ml) dan pindahkan dengan pipet tetes
kedalam tabung reaksi
b) Masukkan secukupnya logam Mg dan beberapa tetes asam klorida pekat.
c) Terbentuknya warna orange sampai merah menandakan adanya flavonoid.
d) Amati dan catat hasilnya.
e) Lakukan percobaan yang sama dengan menggunakan sampel lain.
3. Fenolik
a) Dari fraksi air diambil 3 sampai dengan 5 tetes dan masukkan ke dalam plat tetes
yang bersih. b) Tambahkan 2 tetes pereaksi FeCl3.
c) Terbentuknya warna biru-biru gelap menandakan adanya kandungan golongan
senyawa fenolik
d) Catat hasil pengamatan.
4. Saponin
a) Dari fraksi air, ambil dengan pipet tetes 2 ml lapisan air dan masukkan kedalam
tabung reaksi. b) Tutup mulut tabung reaksi dengan ibu jari, dan kocok dengan kuat
sehingga terbentuk busa. c) Terbentuknya busa yang segera hilang apabila didiamkan
menunjukkan senyawa saponin.
d) Catat hasil pengamatan yang terjadi.
5. Steroid dan Terpenoid
a) Siapakan sebuah pipet tetes bersih, masukkan kedalamnya sedikit kapas dari ujung
bagaian dalamnya, tambahakan arang aktif (Norit) secukupnya.
b) Ambil 1/2 pipet lapisan kloroform dan masukkan hati0hati kedalam pipet tetes yang
sudah dipersiapkan sebelumnya (berisikan norit dan kapas
c) Biarkan kloroform menetes dengan perlahan melalui unjung pipet, apabila tidak
mentes gunakan karet pipet untuk memberikan tekanan sehingga kloroform dapat
menetes keluar.
d) Filtrat kloroform yang menetes harus jernih menandakan norit sudah cukup aktif
untuk mengikat klorofil dan filtrat yang keluar dimasukkan 3 bagian plat tetes, biarkan
sampai mongering
e) Apabila kloroform sudah menguap sempurna dan kering, tambahkan masinh-masing
kedalam.
f) Terbentuknya warna biru-ungu menandakan adanya steroid, sedangkan bila
terbentuknya warna merah menunjukkan adanya kandungan terpenoid.
g) Lakukan percobaan pada sampel yang lain.
h) Catat hasil pengamatan yang dialakukan.

2.8 Hasil Pengujian

 Dari hasil uji fitokimia yang kelompok kami lakukan, dilakukan uji untuk
beberapa sampel tanaman yang didapat dari Lembah Anai, Sumatera Barat, didapatkan
hasil dari kandungan fitokimia tanaman uji sebagai berikut:
Uji Fitokomia Uvaria chamae.:
1. Alkaloid : Positif
2. Fenolik : Negatif
3. Flavonoid : Positif
4. Saponin : Positif
5. Steroid : Negatif
6. Terpenoid : Positif

2.9 Kesimpulan

Penelitian ini menunjukkan bahwa Uvaria chamae memiliki kandungan senyawa

fenolik dan antioksidan yang tinggi aktivitas. Oleh karena itu mereka dapat digunakan
untuk mengobati beberapa penyakit di mana ada peningkatan radikal bebas produksi.
Penelitian ini membenarkan penggunaan tradisional dari akar Uvaria chamae untuk
manajemen disentri di Afrika Barat.

2.10 Saran

Perlu dilakukannya pendalaman pencarian dan validasi lebih lanjut terhadap

komponen komponen fitokimia dari tanaman Uvaria chamae. Menggunakan
instrumentasi untuk memastikkan keberadaan senyawa senyawa metabolit sekundernya
dan potensi dari senyawa tersebut dapat dikembangkan untuk penelitian lebih lanjut
dengan pengujian aktivitas senyawa ekstrak atau isolat dari tanaman ini.
 DAFTAR PUSTAKA

Emeka, E. J., Oluwatoyin, A. E., Adekunle, O. I., & Ignis, I. O. (2015). Preliminary
phytochemical screening and evaluation of hypoglycemic properties of the root
extract of Uveria chamae. Bangladesh Journal of Pharmacology, 10(2), 326–331.
https://doi.org/10.3329/bjp.v10i2.22287

Monon, K., Abdoulaye, T., Karamoko, O., & Adama, C. (2015). Phytochemical
composition, antioxidant and antibacterial activities of root of Uvaria chamae p.
Beauv. (Annonaceae) used in treatment of dysentery in north of Côte d’ivoire.
International Journal of Pharmacognosy and Phytochemical Research, 7(6),
1047–1053.
 Available online on www.ijppr.com
International Journal of Pharmacognosy and Phytochemical Research 2015; 7(6); 1047-1053

ISSN: 0975-4873
Research Article

Phytochemical Composition, Antioxidant and Antibacterial Activities

of Root of Uvaria chamae P. Beauv. (Annonaceae) Used in Treatment
of Dysentery in North of Côte d’Ivoire.
Kone Monon1, Toure Abdoulaye2*, Ouattara Karamoko1, Coulibaly Adama1.

Laboratoire de pharmacodynamie-Biochimique.
1

UFR Biosciences, Université Félix

HOUPHOUET-BOIGNY de Cocody
Abidjan 22 BP 582 Abidjan 22, Côte d’Ivoire.
2
Laboratoire de Biochimie et Sciences des Aliments. UFR Sciences Biologiques, Université Peleforo GON
COULIBALY de Korhogo. BP 1328 Korhogo, Côte d’Ivoire.

Available Online :4th October, 2015

ABSTRACT
In the present work phytochemical composition, antioxidant and antibacterial properties of aqueous and
ethanol extracts of root of Uvaria chamae were studied. Antibacterial activity of extracts of plant was
evaluated on four bacterial strains (E. coli sensitive, E. coli ESBL, S. flexneri ESBL and Shigella sp) by agar
both dilution and agar plate methods. For antioxidant property, free radicals scavenging capacity of the two
extracts were assessed in vitro by 1,1 diphenyl-2picrylhydrazyl (DPPH) radical scavenging and ferric reducing
antioxidant power (FRAP) assays, while inhibition of free radicals generation was assessed by ability of the
extracts to inhibit lipid peroxides formation. Phytoconstituants of two extracts of Uvaria chamae (tannins,
flavonoids, and phenols) were also assayed by colorimetric method. Ethanolic extract of Uvaria chamae
presented bactericidal effect against four strains tested (MBC/MIC = 2). Antibacterial parameters revealed
that among the four bacteria tested, aqueous extract of Uvaria chamae has bacteriostatic power against
ESBL strains (MBC/MIC = 16). The two extracts of plant showed significant (p<0.05) reducing, chelating and
free radical scavenging activities with concentrations required for 50% inhibition (IC 50) of DPPH varying from
3.52±0.38 µg/ml to 14.35±4.86 µg/ml. Ethanolic extract of Uvaria chamae showed significant (p<0.05) high
antioxidant activity (4.02 ± 0.50 μg/ml) than aqueous extract (12.59 ± 2.77 μg/ml). Phytoconstituents
analysis revealed that the two extracts contain significantly (p<0.05) high quantity of total phenols, followed
by flavonoids and tannins. The high presence of phenolic compounds in ethanolic extract could be
responsible of it antioxidant and antibacterial potentiality. The results of these investigations could justify
traditional used of Uvaria chamae in treatment of bacterial dysentery.
 Kone et.al. / Phytochemical Composition…
Keywords: Uvaria chamae, antibacterial, antioxydant, phenolic compound, dysentery, Côte d’Ivoire.
INTRODUCTION
Oxidative stress results from an imbalance between
the oxidant production and a biological system’s
ability to readily detoxify the reactive intermediates
or easily repair the resulting damage1. Increased
oxidative stress is associated with many of the risk
factors implicated in the pathophysiology of
atherosclerosis, including diabetes,
hypercholesterimia, renal failure, aging,
hypertension and smoking2. Reactive oxygen species
(ROS) such as superoxides, peroxides and hydroxyl
radicals are known to play an important role and
have been identified as major contributors to all cell
and tissue damage in many disease conditions3. The
mains endogenous sources of most of the oxidants
produced by cells appear to be normal aerobic
respiration stimulates polymorphonuclear
leukocytes and peroxisomes4. Exogenous sources of
reactive oxygen species include environnemental
pollutants, radiations, antibiotic resistance in
pathogenic organisms and the persistence of
pathogens in immune compromised individuals 5,6.
To protect the cells and organ systems of the body
against reactive oxygen species, the human body
has
*Author for Correspondence
evolved a highly complex antioxidant protection
system. Most synthetic or naturally occurring
antioxidants have phenolic hydroxyl groups in their
structures and the antioxidant properties are
attributed in part to the ability of these natural
compounds to scavenge free radicals7. Several
synthetic antioxidants such as butylated
hydroxytoluene (BHT) and butylated hydroxyanisol
(BHA) are widely used as feed additives to prevent
spoilage. Natural or synthetic antioxidants adverse
effects on human health are still worrying. In order
to find molecules most effective natural
antioxidants and negligible side effects, scientific
research has focused in recent years, its
investigations into new agents of plant origin. In this
IJPPR, Volume 7, Issue 6 : December 2015
context, through an ethno pharmacological
investigation, Koné et al., 2013 showed that Uvaria
chamae, used by the people of northern Côte
d'Ivoire in treatment of dysentery is rich in various
metabolites including polyphenols. Uvaria chamae,
commonly known as finger root or bush banana
belongs to the family Annonaceae. U.chamae is an
evergreen plant which grows to a height of 3.6-4.5
m. Leaves are stipulate, leaf apex 2 cuminate and
the leaf vestiture is glabrous8. It is a small tree
native to the tropical rain forest of West and Central
Africa where it grows in wet and coastal
shrublands9. All parts of the plant are fragrant, the
roots and roots-bark have a widely spread
reputation10. The root (pounded or pulverised) is
used for the treatment of nose bleeding, heart
diseases (bronchi, lungs etc.), and blood in urine,
pile and fever11,12 . These natural substances are
widely distributed in plant species and are
recognized through their beneficial health effects 13.
Their natural antibacterial and antioxidant roles
have more and more interest in the prevention and
treatment of cancer, inflammatory and
cardiovascular diseases14. The secondary
metabolites of medicinal plant could be responsible
for the therapeutic activity attributed to Uvaria
chamae. In order to check the therapeutic benefits
given to this plant, the present study aims to
investigate the in vitro antibacterial activity and
antioxidant property of Uvaria chamae.
MATERIALS AND
METHODS
Chemicals and reagents
Folin-Ciocalteu reagent, sodium carbonate,
methanol, aluminum chloride, potassium acetate,
2,2-diphenyl-1picryl hydrazyl (DPPH), ethanol,
sulfuric acid, potassium dihydrogen phosphate,
potassium ferricyanide, trichoroacetic acid,
ferrosine, iron II chloride, quercetin and gallic acid
were purchased from Ryca-Pharma and Chemical
Laboratory Equipment (CLE), Côte d’Ivoire. All
Page 1048
 Kone et.al. / Phytochemical Composition…
reagents and chemicals used in this study are
analytical grade.
Collection of plant materials
Roots of Uvaria chamae P. Beauv. (Annonaceae)
were colleted from Boundiali Northen Côte d’Ivoire
in September 2010. The plant was identified and
authentificated by Professor Ake-Assi of National
Floristique Center of University Felix Houphouët
Boigny,
Abidjan, Côte d’Ivoire.
Preparation of plant extracts
The roots of plant Uvaria chamae were whased
thoroughly with distillated to remove dirt. After
which root bark were detached and air-dried at
room temperature for five weeks. The dried root
bark were ground well in mechanical grinder; then
passed through the mesh to get uniformly coarse
powder (40 mesh size). This fine powder was stored
in an air tight container. The ethanolic and aqueous
extracts were prepared from 100g powdered plant
material with respectively 400 ml of 70% ethanol
(v/v) and 400 ml of distilled water kept for
maceration for 72 hrs at room temperature. After
which, each extract was filtered twice through
cotton wool, then through Whatman filter paper
N°1 and concentrated by rotary vacuum evaporator
at 45˚C. The two extracts (ethanolic and aqueous)
obtained were dispersed into sterile containers and
stored in a refrigerator for future use. Qualitative
phytochemical analysis
extract were measured spectrophotometrically at
765 nm. The total phenolic content of plant sample
was calculated from the Gallic acid standard curve.
The results were expressed as mg/g of Gallic acid
equivalents16.
Determination of total flavonoids
The content of total flavonoids was done by
aluminum chloride method. In this method the
calibration curve was drawn from different
concentrations of standard quercetin 2 to 10μg/ml
and each extracts of root bark of Uvaria chamae
were prepared. 1 ml of each sample was mixed with
4 ml of distilled water in a volumetric flask and 300
μl of 5% sodium nitrate was added and incubated
for 5 minutes at room temperature. Then 300 μl of
10% aluminum chloride was added to flask and 2 ml
of 1 % sodium hydroxide was added immediately.
The volume is then made up to 10 ml with distilled
water. The absorbance was taken at 510 nm using
UV-visible spectrophotometer. The results are
expressed as mg/g of quercetin equivalent 17.
Determination of total tannins
Tannins contents in each extract were determined
using method described by Baindridge et al., 199622.
1 ml of each sample was mixed with 5 ml of vanillin
reagent (1%) in a test tube. Then the tube was
incubated for 20 minutes in the dark and the
absorbance was taken at 500 nm using UV-visible
spectrophotometer. The results are expressed as
mg/g of tannic acid (2 mg/ml) equivalent.

Qualitative phytochemical analysis of ethanolic and

aqueous extracts of Uvaria chamae was carried out
to identify the secondary metabolites by following
the standard procedures15. Phenolics, flavonoids
and tannins were quantified by the following
methods.
Determination of total phenolics

Different concentrations (20, 40, 60, 80,100 μg/ml)

of Gallic acid standard and two concentrations
50mg/ml and 100 mg/ml of plant extracts were
prepared; 1 ml of each sample and standard were
placed in different test tubes. Then 2.5 ml of a 10%
Folin-Ciocalteu reagent and 2 ml of 2% sodium
carbonate were added and the test tubes were
covered with aluminum foil. This mixture was
incubated for 30 minutes at room temperature and
the absorbance of standard gallic acid and the plant
IJPPR, Volume 7, Issue 6 : December 2015 Page 1049
 Kone et.al. / Phytochemical Composition…
Antibacterial assay
Antibacterial activity of extracts of Uvaria chamae
were performed by broth dilution agar method
coupled with seeding on agar plate. Four strains (E.
coli sensible, E. coli ESBL, S. flexneri ESBL and
Shigella sp) obtained from Bacteriology Laboratory
of Pasteur Institut of Côte d’Ivoire were tested. Each
test compound was incorporated into growth
medium in tubes and Petri dishes to give serial two
fold dilutions. The resulting concentrations ranged
from 3.125 to 100 mg/ml. A tube and Petri dishe
containing nutrient broth only, seeded with test
organism was served as growth control. Bacterial
cell suspensions were inoculated on the tubes and
plates using a bacterial planter (10 µl). All the
inoculated tubes and plates were then incubated at
37°C±2°C for 18 h. The lowest concentration of the
plate, which did not show any visible growth after
was collected from those tubes which did not show
any growth and inoculated on sterile nutrient agar
by streaking. Plates inoculated with bacteria were
then incubated at 37°C for 24 hours. After
incubation the concentration at which no visible
growth was noted as MBC17.
DPPH radical scavenging actvity
Antioxidant activity of the extracts of Uvaria
chamae was assessed through DPPH scavenging
potential with ascorbic acid as the standard 18.
Twenty microliters of various concentrations of
extracts in methanol were added to 1 ml of 0.004%
methanol solution of DPPH. The reaction tubes
were wrapped in aluminum foils and incubated at
room temperature for 1 to 5 min in the dark then,
the absorbance was read at 517 nm. All readings
were recorded in dim light 30. The percent (%)
inhibition of free radical (DPPH) was calculated

Figure 1: Comparison of phytochemical test constituants of ethanolic and aqueous extracts Antibacterial
activity

Table 1 : Composition of phytochemical test constituants of ethanolic and aqueous extracts of Uvaria
chamae

Extracts Total polyphenols (mg GAE/ g) Tannins (mg TAE/ g) Flavonoids (mg QE/ g)
Ethanolic extract 21.14±0.51 9.519±0.96 12.13±1.68
Aqueous extract 8.917±0.92 6.253±1.10 6.833±0.01

incubation, was considered as minimal inhibitory using the formula: Inhibition (%) of DPPH = [(Ac-
concentration (MIC). To determine the minimal As)/Ac] × 100 where, Ac is the absorbance of the
bactericidal concentration (MBC) for each set of test control (containing all reagents except the test
tubes in the MIC determination, a loopful of broth sample/standard) and As is the absorbance of the

IJPPR, Volume 7, Issue 6 : December 2015 Page 1050

 Kone et.al. / Phytochemical Composition…
test sample. Extract concentration providing 50% Statistical analysis
inhibition (IC50) was calculated from the standard
Data were expressed presented and were presented
graph plotted using inhibition percentage against
as mean values ± SD (standard deviations). All the
concentration.
data were
Ferric reducing antioxidant power (FRAP)

The FRAP assay used antioxydants in the redox

linked colorimetric method with absorbance
measured with a spectrophotometer 19. A 300
nmol/l acetate buffer of pH 3.6 (3.1 g of sodium
acetate + 16 ml of glacial acetic acid made up to
one litre with distilled water, 10 nmol/L 1,2,4,6-tri
2pyridyl 1,3,5-triazine, 98%, 3.1 mg/ml in 40 nmol/l
HCl) and 20 mmol/l of ferric chloride were mixed
together in the ratio of 10:1:1, respectively to give
the FRAP working reagent. 1 ml of extract or
standard (vitamin C) was added to 2.5 ml of
KH2PO4- KOH buffer (0.2 mM, pH 6.6) and 2.5 ml of
1% potassium ferricyanide. The mixture was
incubated at 50°C for min. 2.5 ml of 10%
trichloroacetic acid was added to each test tube
before centrifuged at 3000 trs/min for 10 min.
Supernatant was separated and 2.5 ml of this were
mixed with 0.5 ml of 0.1 % iron II chloride. The
mixture was incubated for 10 min at room
temperature and absorbance was measured at 700
nm against a control in same conditions. Extracts
concentrations providing 50% absorbance (EC 50)
was calculated from the standard (vitamin C).
Chelating activity assay

Chelating power of extracts of Uvaria chamae was

measured by spectrophotometric method based on
the dosage of the complex formed by the ferrous
ion (Fe2+) and ferrosine. To 3.7 ml of methanol, 0.1
ml of iron II chloride (2 mM) was added. After 5 min
of incubation, 0.2 ml of ferrosine (5 mM) was
added. Mixture was homogenized and keeps at
room temperature for 10 min before measuring the
absorbance of complexe Fe2+ferrosine at 562 nm
against a control. Different concentrations of EDTA
were used as standard control. Chelating power of
samples was determined by following formula:

Chelating power (%) = [(Acontrol – Asample)/Acontrol] x 100

Extracts concentrations providing 50% absorbance
(IC50) was calculated from the standard (EDTA).

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 Kone et.al. / Phytochemical Composition…
Table 2: Determination of antibacterial parameters of ethanol extracts of roots of Uvaria chamae
Antibacterial parameters of ethanol extracts
Strains MIC (mg/ml) MBC (mg/ml) MBC/MIC Interpretation
Escherichia coli sensible 6.25 12.5 2 Bactericidal
Escherichia coli ESBL 50 100 2 Bactericidal
Shigella sp. 6.25 12.5 2 Bactericidal
Shigella flexineri ESBL 3.125 6.25 2 Bactericidal

Table 3: Determination of antibacterial parameters of aqueous extracts extracts of roots of Uvaria chamae
Antibacterial Parameters of aqueous extracts

Strains MIC (mg/ml) MBC (mg/ml) MBC/MIC Interpretation

Escherichia coli sensible 50 > 100 > 100 -
Escherichia coli ESBL 6.25 100 16 Bacteriostatic
Shigella sp. 100 > 100 > 100 -
Shigella flexineri ESBL 6.25 100 16 Bacteriostatic

Table 4: Antioxidant activity of extracts of Uvaria chamae and vitamin C.

Anti-radical activity Chelating activity Reducing power

Extracts IC 50 (μg/ml) IC 50 (μg/ml) EC 50 (μg/ml)

Aqueous extract 12.59±2.77 11.00±0.01 14.35±4.86
Ethanolic extract 4.02±0.50 6.11±0.55 9.00±0.66
Vitamin C 3.52±0.38 4.30±0.78 6.11±0.90

analyzed by one-way ANOVA and differences
between the means were assessed with
Dunnet/Turkey’s multiple comparison tests.
Differences were considered significant at p < 0.05.
All analyses were carried out using Graph Pad
software, version 5.01 (USA).
RESULTS AND
DISCUSSION
Phytochemical analysis
Table 1 presented composition of total phenols,
flavonoids and tannins of aqueous and ethanolic
extracts of Uvaria chamae. Figure 1 showed
comparison of composition of
these phytochemical compounds of the two
extracts. These
IJPPR, Volume 7, Issue 6 : December 2015
results revealed that botn extracts are rich in these
phytochemicals. However high total phenols
content is noted in relation to flavonoids and
tannins. In addition ethanolic extract has
phytochemical test compounds content (9.51±0.96
µg to 21.14±0.51µg) than the aqueous extracts
(6.253±1.10 µg to 8.917±0.92 µg.). The results of
this study differ from those obtained by Donatus et
al., 200922. For this author, the contents of total
phenols, flavonoids and tannins of extracts of roots
of Uvaria chamae were respectively 0.10±0.30 µg,
5.70±0.03 µg and 0.40±0.03 µg. This concentration
difference can be explained by several factors
including the method used18. Recent studies have
shown the intrinsic factors that may cause this
difference. These are geographical, climatic and
genetic factors but also the degree of maturation
of the plant19.
The MIC values of ethanolic and aqueous extracts
of Uvaria chamae against test strains of bacteria
were shown in Tables 2 and 3. Ethanolic extract
had significantly (p<0.05) stronger activity against
Page 1052
 Kone et.al. / Phytochemical Composition…
Shigella flexneri ESBL with values of MIC (3.125
mg/ml) and MBC (6.25 mg/ml). This extract had
lower level of activity against E. coli ESBL at MIC of
50 mg/ml and MBC of 100 mg/ml. MIC and MBC of
ethanolic extract against E. coli and Shigella sp
were identical with respectively values of 6.25
mg/ml and 12.25 mg/ml. For all strains tested,
ethanolic extract have shown bactericidal activity
according values of MBC per MIC equal to 2.
Aqueous extract had bacteriostatic activity against
the two ESBL strains with MIC of 6.25 mg/ml and
MBC of 100 mg/ml. Antibacterial parameters of
this extract against E. coli sensible and Shigella sp
could not be determined at concentration of 100
mg/ml. However, antibacterial activity of ethanolic
extract of Uvaria chamae was much higher than
the aqueous extract. The results of this study also
indicate that ethanol is a better solvent than water
in the extraction of the active principles of this
plant. This corroborates the reports of Ogbulie et
20 21
al., 2007 and Oguede et al., 2006 that ethanol is
the best solvent for the extraction of most plant
active principles of medicinal importance. The high
antibacterial activity of ethanolic extract can be
attributed to best concentration of phenolic
compounds by this extract. Moreover the work of
Scalbert, 1991 indicate that more phenolic
compounds are oxidized the more they are
inhibitors of microorganisms.
Antioxidant activity Hydroxyl radical scavening activity
Figure 2 show the inhibition curves for the DPPH
radical by aqueous and ethanolic extracts of roots of
U. chamae and vitamin C. Values of IC50 obtained
from these curves (Table 4) show that extracts of U.
chamae have significant (p<0.05) anti-radical power.
Anti-radical activity of ethanolic extract (IC 50 =
4.02±0.50 µg/ml) and that of reference molecule
(vitamin C: IC50 = 3.52±0.38 µg/ml) are almost equal.
Ethanolic extract presented also the best
antioxidant activity among the two extracts of U.
chamae. These results agree with those obtained by
Yéo et al.,201422. These authors in determining the
antioxidant

Figure 2: Inhibition of radical DPPH (%) by extracts of U. chamae and vitamin C.

IJPPR, Volume 7, Issue 6 : December 2015 Page 1053

 Kone et.al. / Phytochemical Composition…

Figure 3: Reducing power of iron by extracts of U. chamae and vitamin C.

Figure 4: Chelating activity of extracts of U. chamae and vitamin C.

activity of several extracts of Cochlospermum planchonii g/ml) is substantially equal to that of the
methanolic extract with ethanolic and aqueous extrats reached similar of Chrysophyllum perpulchrum (IC50 =
4.00 ± 0.288 conclusions. Also, the IC50 of ethanol extract (4.02 ± 0.50 µg/ml) obtained by Bidié et al.,201123.
But greater than

IJPPR, Volume 7, Issue 6 : December 2015 Page 1054

 Bangladesh J Pharmacol 2015; 10: 326-331 1055
24
that of Combretum sp (6.00 ± 0.45 µg/ml) obtained by par N'guessan et al., 2007 .

This interesting activity of ethanoic extract may be due to the richness of this extract at various secondary
metabolites. According to work of N'guessan et al., 200724, there is a correlation between polyphenol content of
extract and anti-radical activity. This justifies the best anti-radical activity of ethanolic extract of U. chamae that is rich
in total phenols. In addition phenolic compounds are recognizing as capable of reacting with free radicals. However, a
synergistic activity between total polyphenols and flavonoids could also contribute to this activity 25.
Reducing power

The curves in figure 3 shows evolution of reducing power of ethanolic and aqueous extracts of roots of U. chamae and
reference molecule (vitamin C). At concentration of 50 mg/ml, the reducing powers of extracts of U. chamae are
significantly (p<0.05) below that of vitamin C. However, based on EC 50 (Table 4) the reducing power of the ethanol
extract (9.00± 0.66 µg/ml) is near to that of the reference molecule (6.11 ± 0.90 µg/ml). Furthermore, EC 50 value of
aqueous extract (14.35 ± 4.86 µg/ml) is almost double of that of vitamin C (6.11± 0.90 µg/ml). The reducing power of
U. chamae is probably due to presence of hydroxyl group in phenolic compounds that can be used as an electron
donor. Indeed antioxidants are considered as reducing and oxidizing inactivators 26. Some previous studies have also
shown that reducing power of a compound is a significant indicator of evaluation of its potential antioxidant
activity27,28.
Chelating activity

The results of study of chelating power of ethanolic and aqueous extracts of U. chamae are presented in figure 4.
Values of IC50 in Table 4 indicate that the ethanolic extract (6.113±0.55 µg/ml) have significantly the high chelating
activity comparatively to aqueous extract (11.0±0.1 µg/ml). This activity is near that of vitamin C (4.307 ± 0.78µg/ml)
and probably due to synergic interaction of phenolic compounds. Among many study of ferric chelating action of
plants extracts that of Le et al., 2007 show that value of IC50 of ethanolic extract of seed of Lycium barbarum
(10mg/ml) is very superior comparatively to ethanolic extract of U. chamae. The chelating capacity of this extract is
very important because of reducing of concentration of transition metals catalysts of lipid peroxidation. Indeed, iron
can stimuli lipid oxidation by Fenton reaction and also accelerates the oxidation decomposing hydroperoxides peroxyl
radicals and alkoxyl wich in turn can sustain the chain reaction.

CONCLUSION
The present study shows that Uvaria chamae has high content of phenolic compounds and high antioxydant activity.
Therefore they can be used to treat several diseases in which there is an increase in free radical production. This study
justified the traditional use of the roots of Uvaria chamae for management of dysentery in West Africa. However,
further studies are needed to identify which phytogenic phenols are responsible for the antioxidant activity of the
roots of plant, and assess the way in which these substances contribute to this activity. In addition to that in vivo
antioxidant and antibacterial assays are needed to confirm the potential use of this plant in the treatment of some
diseases as dysentery.

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 Bangladesh J Pharmacol 2015; 10: 326-331 1058

A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 326-331
Journal homepage: www.banglajol.info
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Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar,
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ISSN: 1991-0088

Preliminary phytochemical screening and evaluation of

IJPPR, Volume 7, Issue 6 : December 2015 Page 1058
 Bangladesh J Pharmacol 2015; 10: 326-331 1059

hypoglycemic properties of the root extract of Uvaria chamae

Emordi Jonathan Emeka1, Agbaje Esther Oluwatoyin2, Oreagba Ibrahim Adekunle2 and
Iribhogbe Osede Ignis1
1
Department of Pharmacology and Therapeutics College of Medicine, Ambrose Ali University Ekpoma,
Nigeria;
2 Department of Pharmacology, Therapeutics and Toxicology, College of Medicine of the University of Lagos, Nigeria.

Article Info Abstract

Received: 19 February 2015
Accepted: 24 March 2015
Available Online: 11 April 2015
DOI: 10.3329/bjp.v10i2.22287
Cite this article:
Emeka EJ, Oluwatoyin AE, Adekunle OI,
Ignis IO. Preliminary phytochemical
screening and evaluation of
hypoglycemic properties of the root
extract of Uvaria chamae. Bangladesh J
Pharmacol. 2015; 10: 326-31.
The purpose of this study is to evaluate the hypoglycemic properties
and preliminary phytochemical screening of Uvaria chamae. The
hypoglycemic properties of U. chamae was assessed on
normoglycaemic rat that received single dose of the extract at 250
and 500 mg/kg body weight and blood glucose levels estimated at 2,
4, and 6 hours (single dose study). The hypoglycemic property of the
extract was also evaluated in normoglycemic rats by oral glucose
tolerance test. Phytochemical screening of the extract for the
presence of secondary metabolites was performed with standard
methods. The extract showed a significant (p<0.05) reduction in blood
glucose levels at 2 and 6 hours compared to control. The oral glucose
tolerance test result also showed a significant decrease (p<0.05) in
blood glucose levels. The study showed that the extract, U. chamae has
hypoglycemic properties which may be accounted for by the presence of the
phytochemicals.

IJPPR, Volume 7, Issue 6 : December 2015 Page 1059

Introduction
Diabetes mellitus has been described as a metabolic disorder of multiple etiologies characterized by chronic
hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin
secretion, insulin action or both (Walter, 1977; Albert et al., 1998; Kumar and Clark, 2005).
This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute

and perform the work . You must attribute the work in the manner specified by the author or licensor.

It is a major degenerative disease in the world today afflicting many lives both in the developed and developing
countries (Mbaka et al., 2012). It is usually irreversible and its late complications result in reduced life expectancy and
major health loss (Kumar and Clark, 2005). Currently diabetes is controlled by diet, exercise, oral hypoglycemic agents
and insulin therapy (Mallick et al., 2007). The high level of treatment failures, unpleasant side effects and enormous
cost associated with oral anti-diabetic drugs have generated an urgent need and desire for alternative treatments
(Suneetha et al., 2010). The preferred choice of plant medicine by many might not be unconnected with the historical
successes recorded in the use of herbal product in traditional system of medicine in managing diabetes mellitus
(Mbaka et al., 2012). Besides, herbal formulations were observed to have fewer side effects and less toxic because of
their rich natural source. Based on these and the support provided for its practice by the World Health Organization,
several scientific investigations are being conducted with the view of identifying new active ingredient of natural
source that would be more effective in the treatment of diabetes mellitus and diabetic complications (WHO, 1980).
Uvaria chamae is a medicinal plant that belongs to the family, Annonaceae. It is a climbing plant commonly found in
West Africa (Irvin, 1961; Okwu, 2004). In this region of the world, it is identified by numerous names such as: Ogholo
by Esan people of Edo state, Ayiloko by the Igalas, Kaskaifi by the Hausas, Oko oja by the Yorubas in Nigeria and
Akotompo by Fulafainte people of Ghana (James et al., 2013). It has been reported to have antivenom activity (James
et al., 2013). The antibacterial and antifungal activities have also been reported (Okwu et al., 2004). However, no
scientific study has been conducted on the anti-diabetic activity of this plant. The aim of this study is to evaluate the
hypoglycemic properties and preliminary phytochemical screening of U. chamae.

Materials and Methods

Plant materials

The roots of U. chamae were obtained from a farm in Uromi, Edo State, Nigeria. They were authenticated by a
taxonomist, mr T.K Odewo, of Botany Department, University of Lagos. The voucher specimen with number LUH 3572
was deposited in the University herbarium.
Preparation of the plant material for extraction

The roots were washed with clean water to remove foreign materials, chopped into small pieces and dried in an oven
at 45 degrees centigrade for four days. They were powdered to coarse particles with electric grinder. The root powder,
weighing 500 g, was extracted with 93.3% aqueous ethanol by maceration with frequent stirring for 5 days. The extract
was filtered using Whatman filter paper No. 4 and concentrated with a rotary evaporator. The concentrated extract
was dried in an oven at 40 degrees centigrade to obtain 22.4 g dry residue (4.5% yields).
Animals

Swiss mice (20-25 g) and Wistar rats (160 ± 20 g) of both sexes were obtained from the Laboratory Animal Center,
College of Medicine, University of Lagos, IdiAraba and were kept under standard environmental condition of 12/12
hours light/dark cycle. They were housed in cages (5 animals per cage), maintained on standard animal pellets (Pfizer
Feeds Plc, Nigeria), and provided with water ad libitum. They were allowed to acclimatize for seven days to the
laboratory conditions before the experiment. The use and care of the animals, and the experimental protocol were in
strict compliance with the Institute of Laboratory Animals Research (ILAR) guidelines on the use and care of animals, in
experimental studies (ILAR, 1996).
Acute toxicity study

The toxicity study was carried out using 35 (male and female) Swiss albino mice (each weighing 20–25 g). The animals
were randomly distributed into a control group and six treated groups, containing five animals per group. After fasting
the animals overnight, the control group was given 0.4 mL of acacia (2%) suspension orally. while each treated group
received oral solution of the extract prepared with 2% acacia in the doses of

1.0, 2.5, 5.0, 10.0, and 15.0 and 20.0 g/kg body weight respectively. The animals were observed continuously for the
first 4 hours and then for each hour for the next 24 hours and at 6 hourly interval for the next 48 hours after
administering the extract to observe any death or changes in general behaviour and other physiological activities (Ayub
et al., 1997; Bürger et al., 2005). The dose that results in 50% mortality (LD 50) was then determined (Figure 1).
Prelimnary phytochemical screening

Phytochemical screen-ing of the extract for the presence of secondary metabolites was performed with standard
methods using the following reagents and chemicals: alkaloids with Mayer’s reagent and Dragendorff’s reagent
(Farns-worth, 1966; Harborne, 1998), flavonoids with the use of 10% lead acetate and 20% sodium hydroxide (Trease and Evans 1983; Sofowora
1993), tannins with 5% ferric chloride solution (Yadav and Agarwala, 2011) and saponins with ability to produce suds (Houghton and Raman,
1998). Terpenes with Liebermann-Buchard test consisting of a mixture of glacial acetic acid and sulphuric acid (Shoppee,
1964). Terpenoids with a mixture of extract and chloroform and concentrated H 2SO4 (Sofowora, 1993).
Assessment of hypoglycemic properties of U. Chamae (single dose study)

Fifteen rats were randomly selected into 3 groups, 5 rats per group. The rats were fasted over-night. Fasting blood
glucose levels of each group was evaluated. Group I, untreated control was given 0.5 mL of 2% Acacia, while Group II
and III were given the extract, orally at doses of 250 mg/kg and 500 mg/kg
respectively. Blood samples were collected for estimation of Blood glucose level from the tail vein at 2, 4 and 6 hours
after giving the extract (Santosh et al., 2007).
 16 CumulativeCumulative
dead alive
Cumulative alive
14

12

10
Cumulative

-2

2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4

Log dose

Figure 1: Determination of LD50 using cumulative dead of the mice and cumulative mice that are alive (Colegate et al., 1993) Effect of the extract on
328 Bangladesh J Pharmacol 2015; 10: 326-331 oral glucose
tolerance test
Table I
Normal rats
Oral acute toxicity of root extract of Uvaria chamae in mice male and
Group Dose (mg/kg) Log dose Mice alive Dead mice Cumulative alive Cumulative dead female were
I 20,000 4.30 0 5 0 15 fasted over
II 15,000 4.18 0 5 0 10 night and
III 10,000 4.0 0 5 0 5 divided into
IV 5,000 3.7 5 0 5 0 four groups of
V 2,500 3.4 5 0 10 0 five rats each.
VI 1,000 3.0 5 0 15 0
Blood samples
Control received 0.4 mL of 2% acacia; Group I, II, III, IV, V, VI: 20,000, 15,000, 10,000, 5,000, 2,500, 1,000 mg/kg respectively; n=5
were collected
from the tail
Table II veins of the
Assessment of hypoglycaemic properties of Uvaria chamae rats to
estimate the
Group Blood glucose levels (mg/dL)
fasting blood
Pre-treatment Post-treatment
glucose levels.
0 (FBG) 2 hours 4 hours 6 hours
Group 1, the
Group I 62.2 ± 2.5 63.8 ± 2.4 53.8 ± 2.5 66.3 ± 7.0 control, was
Group II 57.7 ± 1.6 53.3 ± 1.9a 50.4 ± 1.5 30.4 ± 1.1a given 0.5 mL
Group III 60.8 ± 2.4 52.7 ± 1.5 a
52.1 ± 1.8 36.6 ± 3.3 a
of 2% Acacia
a b
Mean ± SEM; n=5; p<0.05; p<0.01 vs. control group; Group I: control received 0.5 mL of 2% acacia; Group II, III: 250, 500 mg/kg respectively and group 2, 3
and 4 were
given 100, 250 and 500 mg/kg of extract respectively. Thirty minutes after administering the extract, the three groups
were administered 40% (w/v) glucose at a dose of 1 mL/100 g body weight orally (Ogbonnia et al., 2011). Blood
glucose levels monitored at 30, 60 and 120 min intervals and reported as the average glucose level of each group.
Statistical analysis

Analysis of data was done using GraphPad Prism 6. One-way analysis of variance and t-test were used to compare
means. Means ± SEM are shown in all tables. Level of significance was set at p<0.05 or p<0.01.

Results
In the acute toxicity study (Table I), there was no death among the animals that received 1,000-5,000 mg/kg body
weight of the extract. The animals that received 10,000-20,000 mg/kg body weight of the extract died within 24 hours.

160
Control

140 100 mg/kg

120 250 mg/kg

500 mg/kg
100
Cumulative

80

60

40

20

0 30 60 120
Time (min)

The LD50 of the drug was calculated to be 7,080 mg/kg body weight.
Table II shows the hypoglycemic effects of a single oral administration of two doses 250 and 500 mg/kg body weight of
the root extracts of U. chamae in normal healthy rats. These doses showed significant reduction (p<0.05) in blood glucose
levels at 2 and 6 hours compared to control. Rats treated with 250 mg/kg of the extract showed a maximum reduction of 54.2%
in blood glucose level after 6 hours of oral administration. However, the reduction in blood glucose level at the dose of
250 mg/kg body weight at 2 and 4 hours was 16.5 and 6.3% respectively. Also, rats treated with 500 mg/kg of the
extract showed a maximum reduction of 44.8% in blood glucose level after 6 hours of oral administration. The
reduction in blood glucose level at the dose of 500 mg/kg body weight at 2 and 4 hours was 17.4 and 3.2% respectively.
Figure 2 shows the summary of the oral glucose tolerance test. Following oral glucose load in the control group, the
rise in blood glucose level reached a peak at 30 min of glucose load. Decrease in blood glucose level occurred after 30
min but the blood glucose level failed to return to baseline after 120 min. There was a significant decrease (p<0.05) in
blood glucose levels at 30, 60 and 120 min with a percentage decrease of 57.5, 28.6 and 41.4% respectively in the
group of rats treated with 500 mg/kg dose of extract compared with control.

The preliminary phytochemical screening of the root extract of U. chamae revealed the presence of flavonoids,
alkaloids, cardiac glycosides, terpenoid and terpenes, saponin, tannin proteins and sugars (Table III).
Figure 2: Oral glucose tolerance test

Table III
Qualitative analysis of phytochemcal
constituents of root extract of Uvaria
chamae
Compounds Presence
Alkaloids +++
Flavanoids ++
Cardiac glycosides +
Terpenoids and terpenes +
Saponin +
Tannin ++
Protein +
Sugar +
+++ appreciable amount; ++ moderate amount; + trace amount
Discussion
The major goals in the treatment of diabetes has been to keep both short- and long-term glucose levels within
acceptable limits, thereby reducing the risk of long-term complications (Park et al., 2009). This could be achieved by
optimizing both fasting blood glucose and postprandial glucose levels which have been found to be very important in
achieving near normal glucose levels. Postprandial glucose levels have been reported to serve as a better maker of
glycemic control than fasting blood sugar levels (Park et al., 2009).

Drugs that reduce post-prandial hyperglycemia by suppressing hydrolysis of starch have been found useful in the
control of diabetes mellitus (Tundis et al., 2010; Kazeem et al., 2013a). Many herbal extracts have been reported for
their anti-diabetic activities and are currently being used in traditional medicines for the treatment of diabetes.
However, such medicinal plants have not yet gained much importance as medicines due to lack of sustained evidence
(Sudha et al., 2011).

The results of this study showed that the median lethal dose (LD 50) of the root extract was determined to be 7.08 g/kg
body weight translating to 490 g dose for human adult. According to Loomis and Hayes (1996), the extract can be
classified as being practically nontoxic since this value is much higher than Organization for Economic Cooperation and
Development (OECD) toxicity index of 2 g/kg (Walum, 1998; OECD, 2001). Therefore, the extract may be considered to
be safe for consumption.

This study revealed that the rats treated with 250 and 500 mg/kg of the extract had maximum reduction of 54.15 and
44.80% in blood glucose level after 6 hours of oral administration respectively. The extract exerted hypoglycemic
activity by decreasing the blood glucose level significantly. Low blood glucose level reduces the risk of complications
associated with diabetes (Attele et al., 2002; Emordi et al., 2014).

The result of oral glucose tolerance test showed that there was a significant decrease (p<0.05) in blood glucose levels
at 30, 60 and 120 min with a percentage decrease of 57.5, 28.6 and 41.4% respectively in the group of rats treated
with 500 mg/kg dose of extract compared with control.

Insulin release in response to a glucose load occurs in two phases in humans and in rodents. The early phase peaks
within the first 15-30 min and is responsible for limiting the initial rise in glucose upon meal ingestion. The late phase
of insulin secretion occurs later than 30 min after a meal, and may persist for several hours. This delayed burst of
insulin secretion is responsible for returning glucose to baseline fasting levels. In the face of insulin resistance, the late
phase of insulin secretion persists for an extended period and contributes to excessive insulin levels even after a return
to the fasted state, resulting in fasting hyperinsulinemia (Oda, 2012). From this study, the marked reduction in plasma
glucose
330 Bangladesh J Pharmacol 2015; 10: 326-331
concentration
may be as a
result of increased release of insulin from beta cells. This may also account for the hypoglycemic activity of the extract.

The health benefits of medicinal plants are attributed in part to their unique phytochemical composition (Okwu, 2005).
Phytochemicals are secondary metabolites of plant origin which act as antioxidants and stimulate the protective
enzymes in the liver or block damage to genetic materials (Okwu, 2004). They prevent the occurrence of oxidative
chemical species, stimulate antioxidant repairing mechanism and scavenging capa -
city for free radicals in the system.

Flavonoids are phenolic compounds that possess antioxidant and anti-diabetic potentials due to the presence of
hydroxyl groups that confer scavenging ability on them (Mayur et al., 2010). Research has shown that many plants
containing flavonoids have been used for the treatment of diabetes (Meiselman et al., 1976; Choi et al., 1991; Hassig et
al., 1999).
Tannins induce phosphorylation of the insulin receptors as well as translocation of glucose transporters 4 (GLUT-4) ,
the protein factor involved in the signaling pathway of insulin-mediated glucose transport and the inhibition of the
expression of key gene for adipogenesis thereby helping to reduce blood glucose level without increasing adiposity (Liu et al., 2005).

Suba and co-workers reported in 2004 that tannin has anti-diabetic activity.

One of the most important carbohydrate-splitting enzymes is the maltase-glucoamylase which helps to break-down
dietary disaccharides into monosaccharide in the small intestine. If its activity is inhibited, the digestion and absorption
of monosaccharide can be slowed down, decreasing the post-prandial hyperglycemia. The potential therapeutic use of
polyhydroxylated alkaloids in the treatment of type-2 diabetes due to their ability to inhibit maltase-glucoamylase has
been reported (Shang et al., 2013). It is therefore possible that the phytochemicals present in the root extract of U.
chamae may be responsible for the observed hypoglycemic activity.

Conclusion
The LD50 value 7.1 g/kg obtained was a clear indication that U. chamae is safe for use. The study shows that the root
extract of U. chamae has hypoglycemic activity which may be as a result of increased release of insulin from beta cells.

Financial Support
Self-funded

Conflict of Interest
Authors declare no conflict of interest

Acknowledgement
The authors wish to thank Mr. M. E. Idemudia of Idumuune Quarters, Uromi, Edo State, Nigeria, for his assistance in plant collection.

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