INTRODUCTION:
Herbal Medicine sometimes referred to as Herbalism or Botanical Medicine, is the use of herbs for their therapeutic or medicinal value. A herb is a plant or plant part valued for its medicinal, aromatic or savory qualities1. Man has turned to nature for inspiration and guidance from the very beginning of his life on earth to fight against and control diseases. Ages since early human existence, many natural materials by instinct, induction or trial and error got in use for combating human ailments. Thus traditional systems of medicine such as Ayurveda, Siddha, Unani, Homeopathy etc, depend heavily on natural products. However, medicinal plants form the major source of drugs in all the traditional systems of medicines of India. There is a growing importance in medicinal plants and traditional health system in providing health care for a wider population across the globe, especially in the developing countries.
The World health organization (WHO) currently encourages, recommends and promotes traditional remedies in national health care programmes as they are easily available at low cost comparatively safe and are culturally acceptable. Further, WHO estimates that about three quarters of the world’s population currently use herbs and other forms of traditional medicines to treat the ailments2. The plant Mussaenda frondosa Linn belongs to family Rubiaceae, known as white lady in English and Vellila in Malayalam. It is an erect or scandent shrub with grey bark and simple leaves, sometimes a small tree, distributed throughout India in forests, also cultivated in gardens. Bark is grey, leaves simple, opposite, ovate, acuminate at apex, white tomentose beneath and flowers yellowish green outside and orange inside which are found in terminal cymes. One of the calyx lobes become enlarged to white leaf like structure. Fruits globose or ovoid green berries. Roots of the plant are used in leprosy. The juice of the root is used to treat blemishes on the tongue and the sepals are diuretic. Traditionally leaves are used in the treatment of jaundice, asthma, hyperacidity, fever, ulcers, leprosy, diuretic, swellings etc. The antimicrobial activity3, hepatoprotective activity4, activity on fever asthma, wound healing5, hypolipidemic6 and cough was reported in the leaf extract. The plant found to contain Iridoids, Flavanoids and triterpenes, Saponins7 etc. The present study aims the phytochemical screening and in vitro anticancer activities of leaves of Mussaenda frondosa.
MATERIALS AND METHODS:
Plant Collection and Extraction:
The leaves of the plant Mussaenda frondosa linn was collected from Ernakulam district of Kerala India. The plant material was authentified by Dr. V J Dominic, HOD, Department of Botany, Sacred Hearts College Thevara. The leaves collected were dried under shade and then powdered with a mechanical grinder and stored in airtight container. The dried powder material of the leaves (50g) was defatted with petroleum ether and the marc thus obtained was subjected to a successive extraction with chloroform and methanol (250ml). All the extracts finally dried and the traces of solvent were removed by keeping the dried extracts in desiccators. The extracts were stored in a cool place for further study.
Preliminary Phytochemical Investigation:
All the extracts were screened for the presence of various secondary metabolites like flavonoids, glycosides, saponins, tannins, phenolic compounds etc using standard methods8. The powdered crude drug was subjected to determination of Total ash, water soluble ash, acid insoluble ash, moisture content, bitterness value, swelling index and foaming index.
Isolation of total chemical constituents from methanolic extract9:
Stationary phase:
5x10 cm and 20x10 HPTLC silica gel 60 F254 (Merck) plates.
Sample application:
About 9-40 µL of methanolic extract of leaves of plant was spotted with a band width of 8 mm, each spot at least 2 mm apart.
Solvent Systems Used:
Chloroform: ethylacetate (7.5:2.5) for flavanoids.
Ethylacetate: 1, 4-dioxane: formic acid: water (5:3:1:1) for phenolic compounds.
10x10 cm Twin Trough Chamber was used and solvent system prepared was saturated for 20 min.
METHOD:
A 9µL of methanolic extract was spotted in 5x10 cm HPTLC silica gel 60 F254 (Merck) plates and screening was done. For isolation of chemical constituent 16 spots each of 40µL of methanolic extract was spotted in 20 x 10 cm HPTLC silica gel 60 F254 (Merck) plate and was developed using specific solvent system. Then the region between the solvent front and sample application which contain the total chemical constituent was scratched out and the powder was divided into 2 portion and to each 10ml methanol was added and sonicated for 10 minutes. Then the samples were centrifuged at 2000rpm for 10minutes.The obtained solution is filtered and concentrated and stored in a dry container10.
In vitro cytotoxic activity:
Cell lines and Culture medium:
HepG2 (Human liver hepatocellular carcinoma cell line), was cultured in DMEM supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B (5 mg/ml) in an humidified atmosphere of 5% CO2 at 37°C until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures were grown in 25 cm2 culture flasks and all experiments were carried out in 96 microtitre plates.
Preparation of Test Solutions:
For cytotoxicity studies, each weighed test drugs were separately dissolved in distilled DMSO and volume was made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two fold dilutions were prepared from this for carrying out cytotoxic studies.
Procedure:
The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 x 105 cells/ml using DMEM containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 ml of the diluted cell suspension (approximately 10, 000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was flicked off, washed the monolayer once with medium and 100 ml of different test concentrations of test drugs were added on to the partial monolayer in microtitre plates. The plates were then incubated at 37oC for 3 days in 5% CO2 atmosphere, and microscopic examination was carried out and observations were noted every 24 h interval. After 72 h, the drug solutions in the wells were discarded and 50 ml of MTT in PBS was added to each well. The plates were gently shaken and incubated for 3 h at 37oC in 5% CO2 atmosphere. The supernatant was removed and 100 ml of propanol was added and the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (CTC50) values is generated from the dose-response curves for each cell line11
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% Growth Inhibition = 100- |
Mean OD of individual test group |
X 100 |
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Mean OD of control group |
RESULTS AND DISCUSSION:
The powdered crude drug was subjected to determination of total ash, water soluble ash, acid insoluble ash, moisture content, bitterness value, swelling index and foaming index. These values are useful in determining the quality and purity of crude drug. Further these values indicate the nature of the constituents present in a crude drug.
Table-1: Physicochemical Parameters
|
Parameters |
Percentage value |
|
Moisture Content |
16% |
|
Total Ash |
13%w/w |
|
Acid Insoluble Ash |
4.5%w/w |
|
Water soluble Ash |
7.5%w/w |
|
Bitterness Value |
1700 |
|
Foaming Index |
100 |
|
Swelling Index |
2.30cm |
The phytochemical tests revealed that the leaves of Mussaenda frondosa Linn possess presence of various secondary metabolites like flavonoids, glycosides, saponins, tannins, phenolic compounds as shown in Table-2. Since the methanolic extract shows higher concentration of chemical constituents it was subjected for the further study.
Table-2 : Preliminary Phytochemical Screening
|
Sl no |
Chemical test |
Petroleum ether extract |
Chloroform extract |
Methanol extract |
|
1 |
Alkaloids |
- |
+ |
- |
|
2 |
Glycosides |
- |
+ |
- |
|
3 |
Phenolic compounds |
- |
+ |
+ |
|
4 |
Flavanoids |
- |
+ |
+ |
|
5 |
Tannins |
- |
- |
+ |
+ Indicates present, - Indicates absent
The isolation of total flavonoids and phenolic compounds were done using HPTLC as shown in Figure 1.
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Flavonoids 366nm |
254nm |
366nm |
254nm |
|
Flavonoids |
Phenolic compound |
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Figure-1 Isolation of Flavanoids and phenolic compounds