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Isolation, Characterization and Cytotoxic Potential of Desmodium oojeinensis (Roxb) H. Ohashi: A Threatened Medicinal Plant

Desmodium oojeinensis, which belongs to the family Fabaceae, is an endangered plant native to the Himalayan and sub-Himalayan tract. The current work aimed to carry out physicochemical, phytochemical screening, isolation, and in vitro cytotoxic activity using MCF-7 and A-549 cell lines by using the sulforhodamine B method. The physicochemical parameters tested were found to comply with pharmacopoeial limits. The extract’s phytochemical screening revealed triterpenoids, alkaloids, anthraquinone glycosides, flavonoids, and carbohydrates. Four phytoconstituents belonging to the class of triterpenoids, viz. betulin, betulinic acid, 16-hydroxybetulin, and lupeol, were isolated from the ethanolic extract by column chromatography. The ethanolic extract showed moderate cytotoxic activity on the human lung cancer cell line A-549 at a concentration of 80 μg/ml and growth inhibition of 81.5%. The extract failed to hinder the growth of the human breast cancer cell line MCF-7.


INTRODUCTION
Medicinal plants have proven to be potent allies since time immemorial by curbing various health ailments. Although synthetic drugs have substituted herbal healing to a certain level, the resurgence and attentiveness of herbal medicines are returning (Acharya and Shrivastava, 2008). Medicinal plants are affordable and ecofriendly, but possess (Tridevi, 2008) fewer side effects than synthetic drugs. Among the modern drugs in use today, about 40% are of natural origin. Approximately 60% of anti-cancer remedies and 75% of drugs for infectious diseases are natural or derivatives (Samuelsson and Bohlin, 2009). Among the numerous plants available in nature, few of them have attracted scientists' interest to investigate cancer treatment. Phytoconstituents have played a crucial role in developing leads, which are proved to be clinically valuable in treating neoplasm (Shah B et al., 2010).
Desmodium oojeinensis (Roxb) H. Ohashi is a medicinal plant that belongs to the family Fabaceae. It is commonly known as sandan, Tinsa in Hindi, and Ratha in Sanskrit. It is a deciduous tree that is distributed among the Himalayan tracks up to an altitude of 1,500 mts and spread across the whole of northern and central India. Traditionally, it is used for various ailments, such as an astringent, stimulant, anti-inflammatory, urinary astringent, acrid anthelmintic, cooling, and rejuvenating (Kirtikar and Basu, 2006;Nadkarni, 1976). Scientifically, the extract from various parts of the D. oojeinensis plant were screened for anti-inflammatory, analgesic, antispasmodic, hepatoprotective, wound healing, and antimicrobial activities (Khare, 2004;Mandrekar et al., 2014;Sahu and Roy, 2009;Sahu et al., 2008Sahu et al., , 2009. Phytoconstituents, namely genistin, kempferol, lupeol, butuline, hydroxylupeol, and isoflavones, like dalvergioidin, homoferreirin, and Eugenie, from various parts of the plant have been reported (Balakrishna et al., 1962;Ghosh and Dutta, 1965;Mukherjee et al., 1963).
The current work aimed to accomplish physicochemical, phytochemical screening, isolation, and in vitro cytotoxic activity using MCF-7 and A-549 cell lines by using the sulforhodamine B (SRB) method.

Collection and authentication
The fresh bark of the stem was collected from fully grown D. oojeinensis (Roxb) H. Ohashi plants from Chitradurga, Karnataka, India. It was authenticated by Dr. K. Gopalkrishna Bhat, Department of Botany, Poornaprajna College, Udupi. The archived specimen is banked at Goa College of Pharmacy (GCP/ Ph.cog 2019/P001) for future reference.

Physicochemical parameters
Various physicochemical parameters, such as swelling index, moisture content, foaming index, ash values, and extractive values, were determined as per the World Health Organization's guidelines (Anonymous, 1998).

Extraction and preparation of extract
Coarsely powdered 400 g of air-dried stem bark was extracted with ethyl alcohol (95%) for a period of 3 days at room temperature. The ethanolic layer was decanted off. The operation was repeated thrice. By the rotary vacuum evaporator, the solvent was recovered, and the residue was evaporated and concentrated to a syrupy consistency and then dissipated to dryness (4.25% w/v).

Preliminary phytochemical screening
Preliminary phytochemical studies were carried out on the ethanolic extract of the bark of the stem of D. oojeinensis to check for the presence of chemical constituents like alkaloids, carbohydrates, glycosides, flavonoids, triterpenoids, steroids, tannins, phenolic compounds, proteins, resins, starch, etc. (Khandelwal, 2010;Kokate et al., 2006;Shah and Seth, 2010).

In vitro cytotoxic activity
In vitro cytotoxic activity of the ethanolic extract of D. oojeinensis was executed by SRB assay. The sample was prepared in dimethyl sulfoxide (DMSO) to obtain the concentration of 100 µg/ml. Human breast cancer cell lines, MCF-7, and human lung cancer cell line, A-549, were seeded at a density of 10 3 per well in a 96-well Petri plate. The plates were incubated after the addition of samples at concentrations of 10, 20, 40, and 80 µg/ ml and incubated at 37°C for 48 hours in a 5% CO 2 incubator. The assay was ceased by the addition of cold trichloroacetic acid (TCA). 30% w/w TCA (50 µl) was used to root the cells in situ and incubated for 60 minutes at 4°C. The supernatant was rejected, and the plates were rinsed with tap water, and allowed to air-dry. All the wells were supplemented with 50 µl SRB solution (0.4% w/w in 1% acetic acid). The plates were incubated for 20 minutes at 27°C. 1% w/w acetic acid was used to remove unbound dye, and the plates were dried. The bound stain was eluted with 10 µM Trizma base and absorbance was taken at 540 nm. The values were recorded in triplicate and compared with the standard adriamycin (10-80 µg). Growth (%) was calculated on the plate-by-plate basis for tested wells compared to control wells. Growth (%) was articulated using the following formula: Average absorbance of test wells × 100 / Average absorbance of control wells (Houghton et al., 2007;Lamkanfi et al., 2008).

Statistical analysis
Mean ± standard deviation (S.D.) was used to express the values.

RESULTS AND DISCUSSION
The results of physicochemical and phytochemical investigations are depicted in Tables 1 and 2, respectively. The physicochemical investigation of the powdered bark of the stem of D. oojeinensis was found to be within the limits of pharmacopoeial standards. A preliminary phytochemical investigation of the ethanolic extract disclosed the existence of alkaloids, carbohydrates, flavonoids, triterpenoids, steroids, tannins, phenolic compounds, and anthraquinone glycosides.

In-vitro cytotoxic study
The cytotoxic study results on human lung cancer cell lines, A-549, and human breast cancer cell lines, MCF-7, are given in Tables 3 and 4. The result indicates moderate cytotoxic activity against A-549 cell lines at 80 µg/ml with growth inhibition of 81.5% when compared to the standard adriamycin with percentage growth inhibition of <10 µg/ml. The extract failed to show in vitro cytotoxic activity against MCF-7 cell lines. The majority of pentacyclic triterpenoids have proved to possess cytotoxic activity. Betulin and betulinic acid exhibit effective anti-cancer activity by activating the mitochondrial apoptosis pathway in tumor cells (Hordyjewska et al., 2018). Chemical investigation led to the isolation of terpenoids having lupine moiety, isolated from the extract of D. oojeinensis, which may be responsible for moderate cytotoxic activity against human lung cancer cell lines A-549.

CONCLUSION
The current research work on the bark of the stem of D. oojeinensis was successfully explored. The physicochemical parameters tested were found to be within the pharmacopoeial limits. Chemical examination of the extract was directed to the separation of four triterpenoids, namely betulin, betulinic acid, 16-hydroxybetulinic acid, and lupeol. Based on a literature survey, betulin and betulinic acid exhibited significant cytotoxic activity. The ethanolic extract showed moderate cytotoxic activity in A-549. The activity might be endorsed due to betulin and betulinic acid, which have been isolated from D. oojeinensis. Apoptosis and in vivo studies will further potentiate its claim as an effective cytotoxic agent in lung cancer treatment.