INTRODUCTION:

Tylophora indica (Burm f.) Merill. (Family: Asclepidaceae) commonly known as Damvel or Antmool is a perennial plant distributed throughout southern, western and eastern part of India in plains, forests, and hilly places [1]. The plant is found growing normally in Maharashtra, Gujrat, Uttar Pradesh, Bengal, Karnataka, Assam, Orissa, Himalayas and sub Himalayas in India [2]. It is a branching climber that grows up to 4.5 feets, leaves are obvate-oblong to elliptic-oblong, 3-10 cm long and 1.5-7cm wide [3]. The T.indica has been reported to contain 20.46% alkaloids viz. Tylophorine, tylophorinine, tylophorinidine, (+) septicine, isotylocrebrine, tylophorinicine, sterols, flavonoids, wax, resins, and tannins [5].

The T.indica has been used in Ayueveda as traditionally used for the treatment of bronchial asthma, jaundice and inflammation [3,6]. The properties like antitumor, immunomodulatory, antioxidant, anti-asthmatic, smooth muscle relaxant, hypotensive, antirheumatic activities are scientifically proven. In Ayurveda, the plant has been used in treatment of asthma, dermatitis and rheumatism [1,6]. Although the leaves and root of this plant are widely used for treating jaundice in Northern Karnataka and Maharashtra, there is a paucity of scientific evidence regarding its usage in liver disorder [3]. The other reported activities include immunomodulatory activity, anticancer activity, anti-inflammatory activity and anti-amoebic activity [7,8,9,10].

Botanical name: Tylophora indica (Burm f.) Merrill.

Synonym: Tylophora asthmatica (Linn. F.).

Common Name: Antmul.

Other names: Bengal- Antomul.

Konkan and Maharashtra- Pitmari, Kharaki- raena, Anthamul, Pitakari.

Gujrat- Antamul.

Hindi- Antamuli.

Kannada- Kirumanji.

Malayalam- Valli-pali.

Marathi- Pitakari, Khodki-Rasna.

Orissa- Mendi, Mulini.

Tamil- Naye-pallai.

Telangana- Veripala, Kukka-pala, Vettipala [4].

Habitat:

The plant is a biannual perennial branching climber with long leaf and fleshy roots. It grows in planes and hilly places of India up to an attitude of 1000-1200 m in Maharashtra, Bengal, Assam, Orissa, western and southern India [11].

Geographical Source:

It is traditional or Indigenous to India. The plant inhabits up to an elevation of 1,260m in the sub Himalayan tract and in central, western and in peninsular India. It also met within Eastern, North- East and Central India, Maharashtra, Gujrat and parts of South India [4].

Chemical constituents:

The active constituents of Tylophora indica are alkaloids like tylophorine, tylophorinine, tylophorinidine and septidine. Phytochemical Studies The drug Tylophora indica contains major chemical constituents Tylophorine, α-amyrin, kaempferol, and quercetin, other major alkaloids like tylophorindine, desmethyltylophorine, desmethyltylophorinine, dehydrotylophorine, desmethyltylophoridine, anhydrousdehydrotylophorinine. Other alkaloids (+)-isotylocrebrine (+) Septicine from fresh leaf [4]. Tylophorine B possess a cleaved substituted phenanthrene moiety nucleus and an angular methyl group on the indolizidine portion [12]. Although the potency of structural analogue for cytotoxicity, selectivity against NF-kB signalling pathway, and their inhibitory effects against protein and nucleic acid synthesis are different. Because they don’t have an identical spectrum of targets, the studied are structural but may not be functional analogues [13].

Pharmacological studies

Hepatoprotective activity:

The alcoholic extracts of T. indica leaves was screened for hepatoprotective activity in CCL₄ induced hepatotoxicity in albino rats. T. indica leaves exhibited significant reduction in serum hepatic enzyme when compared to rats treated with CCL₄ alone [14]. The hepatoprotective activity of alcoholic and aqueous extracts of leaves of T. indica against ethanol-induced hepatotoxicity. Ethanol induced significant changes in physical, biochemical, histological, and functional liver parameters. Pre-treatment with ALLT and AQLT extract significantly prevented the physical, biochemical, histological and functional changes induced by ethanol in the liver [14].

Antiallergic activity:

The anti-allergic effect of T. indica was compound with that of disodium cromoglycolate on perfused rat lung in sensitized rats by observing the changes in the volume of the perfusate per minute. Administration of aqueous extract of T. indica and disodium chromoglycolate during perfusion of sensitized rat lung significantly increased the rate of flow. The action of T. indica may be due to direct bronchodilator property and membrane stabilising and immune-suppressive effects [15].

Immunomodulatory activity:

Studies with T. indica alkaloids had shown that they inhibit cellular immune response like contact sensitivity to delayed hypersensitivity to sheep red blood cells, in vivo. The alkaloids mixture suppressed Inter Leukin-2 production at the lower concentrations. Inter Leukin-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentration dependent biphasic effect on con A induced mitogenesis. Crude extract of the leaves of T. indica inhibited delayed hypersensitivity reaction to sheep red blood cells in rats when the alkaloid mixture was administered before and after immunization with these cells.

The alkaloid mixture inhibited contact sensitivity to dinitro-fluorobenzene in mice when given prior to or after contact sensitization. Lymphocytes taken from contact sensitized mice, when treated with tylophora alkaloid in vitro and transferred into naive syngeneic hosts, could suppress the transfer of delayed type hypersensitivity (DTH) response. However, the tylophora alkaloids could not suppress primary humoral (IgM) immune response to SRBC in mice at the same dose. These studies suggest that T. indica alkaloids suppress cellular immune responses when administered at any stage during the immune response [16].

Anti-Cancer Activity:

Tylophorine not only retards the S-phase progression but also dominantly arrests the cells at G1 phase in HepG2, HONE-1, and NUGC-3 carcinoma cells. Moreover, the tylophorine treatment results in down regulated cyclin A2 expression and overexpressed cyclin A2 rescues the G1 arrest by tylophorine. Thus, we are the first to report that the downregulated cyclin A2 plays a vital role in G1 arrest by tylophorine in carcinoma cells [17].

Antimicrobial and Antifungal activity:

Crude and pure extracts of T. indica were investigated in view of antimicrobial activity. Pure compounds displayed strong antibacterial activity at lower concentrations in all tested bacterial strains except E.coli. while all the crude and pure compounds showed antifungal activity against Aspergillus niger, Aspergillus fumigates and Trichoderma virdae, the pure compounds had strong antifungal activity compared to crude extracts [18].

Anti-Asthmatic:

A brief exposure of human peripheral white blood cells from asthmatic children to tylophorine (an alkaloid occuring in T. asthematica) caused the stimulation of adenyl cyclase. This effect was not observed in the leukocytes from the non-asthmatic children or adults [19].

MATERIALS AND METHODS:

The present chapter describes the procedures followed for obtaining Tylophora indica leaves extracts to study its effect on carbon tetrachloride induced hepatotoxicity model of rats.

Location of study:

The present study was undertaken at the Department of Veterinary Pathology, Mumbai Veterinary College, Parel, Mumbai and Department of Veterinary Pharmacology and Toxicology, COVAS, Udgir Dist. Latur (MH). Experimental work to monitor the effect of ethanolic extracts of Tylophora indica

Preparation of plant extract:

The plant Tylophora indica was collected from “Maharashtra Nature Park, Mahim, Mumbai” and the leaves of the plant were collected from “Directorate of Medicinal and Aromatic Plants Research: Anand of Gujrat state, India”. The leaves were shed dried in departmental room. The plant under study was also submitted at Blatter Herbarium of Department of Botany, St. Xavier’s College, CSMT, Fort, Mumbai. In the laboratory, fresh leaves of Tylophora indica were washed with clean water and allowed to dry completely under shade. Dried leaves were ground to powder with the help of an electrically operated grinder. The powered material (leaves) was subjected to 70% ethanolic extraction by batch extraction method (FIG. 1)

Preliminary phytochemical analysis:

The 70% ethanolic extract of T. indica leaves plant material was subjected to preliminary phytochemical screening as per Sanmugarajah [20], at College of Veterinary and Animal Sciences, Udgir, Dist. Latur, Maharashtra and it revealed the presence and absence of various phytochemical constituents, as under.

Determination of alkaloids:

Following two tests were employed for determination of alkaloids in both the extracts prepared us under.

Dragendroff’s Test:

Extracts were dissolved individually in dilute Hydrochloric acid and filtered. Filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide).

Inference:

Formation of red precipitate indicates the presence of alkaloids.

Mayer’s Test:

One ml portions of each extract was acidified with 2-3 drops of 1M Hydrochloric acid and treated with 4-5 drops of Maye’s reagent (Potassium Mercuric Iodide.

Inference:

Formation of a yellow or white coloured precipitate or turbidity indicates the presence of alkaloids.

Determination of flavonoids:

Shinoda test:

The extracts were dissolved in methanol (50%, 1-2ml) by heating. To an alcoholic solution of each of the extract, three pieces of magnesium chips were added followed by a few drops of concentrated hydrochloric acid.

Inference:

Appearance of an orange, pink or red to purple colour indicates the presence of flavonoids.

Determination of glycosides:

Molisch’s test:

Two ml portion of each extracts were shaken with 10 ml of water, filtered and filtrate was concentrated. To this 2-3 drops of Molisch’s reagent was added, mixed and then 2ml of concentrated sulfuric acid was placed carefully through the side of the test tube.

Inference:

Reddish violet ring appear, indicating the presence of glycosides.

Determination of carbohydrates:

Benedict’s test-

Extracts were shaken with 10ml of water, filtered and filtrate was concentrated. To this 5ml of Benedict’s reagent solution was added and boiled for 5 minutes.

Inference:

Formation of brick red coloured precipitate indicates the presence of Carbohydrates.

Anthron test:

Extract was shaken with 10ml of water, filtered and filtrate was concentrated. To this 2ml of anthron reagent solution was added.

Inference:

Formation of green or blue colour indicates presence of carbohydrates.

Detection of proteins:

Xanthoproteic Test:

The extracts were treated with few drops of conc. Nitric acid.

Inference:

Formation of yellow colour indicates the presence of proteins.

Detection of reducing sugar:

Fehling’s test:

To a test tube 1 ml each a Fehlin’s A and B solutions were added and mixed. To this 2 ml of plant extract was added and heated on a boiling water bath for 10 minutes.

Inference:

Formation of brick red or orange precipitate indicates the presence of reducing sugar/ carbohydrates.

Determination of saponins:

Foam Test:

0.5 ml of each extracts were shaken with 2ml of water.

Inference:

Foam produced persists for ten minutes indicates the presence of saponins.

Determination resins:

Acetone Test:

One ml of each extracts was dissolved in acetone and the solutions were poured in distilled water.

Inference:

Turbidity indicates the presence of resins.

Determination of steroids:

Salkowski’s reaction:

Each extracts were shaken with chloroform, to the chloroform layer sulphuric acid was added slowly by the side of test tube.

Inference:

Formation of red colour indicated the presence of steroids.

Determination of tannins:

Ferric chloride test:

A small quantity of the each extracts were boiled with water and filtered. Two drops of ferric chloride was added to the filtrate.

Inference:

Formation of a blue-black or green blackish colour in the presence of ferric chloride precipitate was taken as evidence for the presence of tannins.

Determination of triterpenoids:

Sulphuric acid test

Two mg of dry extract was dissolved in acetic anhydride, heated to boiling, cooled and then 1ml of concentrated sulphuric acid was added along the side of the test tube.

Inference:

Formation of a pink colour indicated the presence of triterpenoids.

RESULTS AND DISCUSSION:

The present study was designed to evaluate the extractability and preliminary photochemical analysis of hydroalcoholic extract of Tylophora indica leaves. The results obtained in present investigations are interpreted and discussed in the present chapter.

Authentication of herbs under study:

The plant Tylophora indica was collected from “Maharashtra Nature Park, Mahim, Mumbai” and the leaves of the plant was collected from “Directorate of Medicinal and Aromatic Plants Research: Anand of Gujrat state, India”. The leaves were shed dried in departmental room. The plant under study was also submitted at Blatter Herbarium of Department of Botany, St. Xavier’s College, CSMT, Fort, Mumbai and was identified and authenticated as Tylophora indica.

Extractability (W/W):

The course powder of plant material was subjected to Soxhlet’s Extraction Apparatus using 70% ethanol and later proceed for evaporation of colourless solvent obtained on water bath to get semisolid material. The 220gram crude ethanolic extract of Tylophora indica was obtained from 700gram material. Therefore, by calculation percent extractability of the Tylophora indica material in 70% ethanol was found to be 31.42%.

Phytochemical screening:

The plant kingdom has lots of hidden secrets, such as their complex compounds or active principles present in it which is thought to be responsible for their therapeutic efficacy. Current investigation was mainly aimed to evaluate chemical composition and phytoconstituents present in the plants under study to standardize the herbal drug under research.

The 70% ethanolic extract of Tylophora indica plant material was subjected to preliminary phytochemical screening as per procedure cited by Sanmugarajah [20] at College of Veterinary and Animal Sciences, Udgir, Maharashtra and it revealed the presence or absence of various phytochemical constituents (Qualitative) as depicted in table below.

Table 1: Results of phytochemical screenings (Qualitative) of extract of the plant material (Fig. 1)

Sr. No.	Chemical Constituent	Test applied	Ethanolic extract
1)	Alkaloids	Dragondroffs	Present
1)	Alkaloids	Wagners	Present
2)	Flavonoids	Shinoda	Present
3)	Carbohydrates	Anthron	Present
3)	Carbohydrates	Benedicts	Present
4)	Proteins	Xanthoprotein	Absent
4)	Proteins	Biuret	Absent
5)	Phenolics	Ferric chloride	Present
6)	Saponins	Foam	Present
7)	Resins	Acetone	Absent
8)	Steroids	Sulkowitch	Present
9)	Tannins	Ferric chloride	Present
9)	Tannins	Lead-acetate	Present
10)	Reducing sugars	Fehling’s	Present
11)	Glycosides	Molisch’s	Present
12)	Triterpenoids	Sulphuric acid	Present

The present study includes phytochemical (qualitative) studies in Tylophora indica plant extract (Ethanolic) was carried out for alkaloids, flavonoids, Glycosides, phenols, saponins, Proteins, steroids, tannins, and Resins All of the phytochemicals like alkaloids flavonoids, steroids, phenols, Tannins, saponins and terpenoids were present in T. indica. Alkaloids are however reported in the present study which agrees with the findings of [21] who has attributed analgesic, antispasmodic and bactericidal effects.

The present study also reports Saponins, similar to the report of [22]. Alkaloids and Saponins are known to be effective for the treatment of syphilis and other venereal diseases, had earlier reported that Saponins have antibiotic properties and so help the body to fight infections and microbial invasion. Also, it is used as a mild detergent and in intracellular histochemistry staining to allow antibody access to intracellular proteins. These proteins were also reported in hypercholesterolemia, hyperglycemia, antioxidant, anticancer, anti-inflammatory and weight loss and have anti-fungal properties [23] reported the presence of tylophorine alkaloids in Tylophora asthamatica.

Investigation of Tylophora indica for the presence of tylophorine is therefore needed in exclusive studies The presence of flavonoids are reported in Tylophora indica presently which is in agreement with [22] who also reported the diuretic property of extracts of Tylophora indica is very valuable information. The present study which agrees with the findings of [24] and [25] reported about medicinal plants.

CONCLUSIONS:

From present investigations it is observed that the phytoconstituents like viz. Alkaloids, Flavonoids, Carbohydrates, Phenolics, Saponins, Resins, Steroids, Tannins, reducing sugars, Glycosides and Triterpenoids are present in the extract. However, the percent extractability of the Tylophora indica material in 70% ethanol is 31.42%.

ACKNOWLEDGEMENTS:

The authors are thankful to the, MAFSU, Nagpur, India, for providing support and the necessary facilities for the research work.

CONFLICT OF INTEREST:

There are no conflicts of interest.

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Received on 03.02.2020 Modified on 12.02.2020

Res. J. Pharmacognosy and Phytochem. 2020; 12(1):. 01-06.

DOI: 10.5958/0975-4385.2020.00001.1