Phytochemical analysis, antioxidant and anti-inflammatory activity of leaves and bark of Ceropegia

Shubhada S Nayak1, Nitin A Mirgane2, K B Pathade3, Vitthal S Shivankar4 & Gurumeet C Wadhawa*1 1Rayat Shikshan Sansthas, Karmaveer Bhaurao Patil College, Vashi, Navi Mumbai, MH, India 2SIES College of Arts, Science and Commerce, Sion (West), Mumbai 400 022, MH, India 3Maharaja Jivajirao Shinde ASC College, Shrigonda, Dist. Ahmednagar 413 701, MH, India 4Rayat Shikshan Sansthas, Chhatrapati Shivaji College, Satara, MH, India *Email: wadhava.gurumeet@gmail.com


Introduction
Inflammation is an important adaptive rejoinder caused by noxious stimuli and conditions such as tissue injury to the animal. Mostly the oxidative stress and inflammatory establish a complex interaction. The endocellular state shows a key role in the activation and dysfunction of various immune cell (1)(2)(3). Ceropegia rollae Hemadri shows significant radical scavenging activity and also interact with various cell lines (4)(5)(6).
Free radicals are highly reactive species; they may have one or more than one unpaired electrons. Such as the superoxide, hydroxyl and peroxy radicals. They may easily attack the cell lines and convert the normal cell into pathogenic cell. These free radicals are responsible for the large nobler of the diseases in the human and the animals. Due to increase in the population and the industrialization, the free radicals are increased in the atmosphere. These free radicals affect the human life badly (6,7). To overcome this, there is need of antioxidants. This antioxidant plays important role for the protection of the body against damage by the radicals. These free radicals are highly reactive. Oxygen radicals are called as ROS and produced by industrial pollution or they are formed by the biochemical process from various cells and the respiratory chain. These free radicals are the main factor for the lipid peroxidation. Ceropegia rollae has certain bioactive compounds, which have good antioxidant and anti-inflammation activities (8)(9)(10).
The plant Ceropegia rollae play an important role in inhibition of the various bacterial disorders and overcome the free radical activity and the inflammation. The phytochemicals present in the plant Ceropegia rollae are the tannins, terpenoids, steroids, phenols, cardiac glycosides, saponins, flavonoids, alkaloids and anthraquinones, determined by various protocols developed (11)(12)(13). They also give positive test for the flavonoids from the Mg-HCl along with the Zn-HCl (14,15). The plant also gave Keller and Killani test for the glycosides and with the acetic anhydride and sulphuric acid for the, ferric chloride as reagent test for the phenols (16,17).
Ceropegia rollae is a tuberous herb, which is widespread to Western Ghats and Konkan region of Maharashtra, India. The Stem is pubescent, leaves are ovate, puberulous, cymes in subaxillary and terminal. Flowers are larger, with peduncle and having pedicals hirsute in nature. The flower is 2.3-3.5 cm long, tube with length 1.5-2.5 cm long, base slightly the inflated at all angles. Mostly the flower Petals are 8-16 x 2.8 mm, linear, oblong with straight, hairless. (18)(19)(20)(21). Flowers are ciliate. They are inner erect, subclavate in nature. Flowering is in the month of August-September.
There are around 200 species of the Ceropegia, among which 58 species are endemic to the Western Ghats of Maharashtra, India (22).
In the present study, we have used the leaves and the bark of the plant. The bark and leaves shows good anti-inflammatory and anti-oxidant activity.

Chemicals and Reagents
The chemicals used were of analytical grades. All chemicals were purchased from the S.D. Fine and Loba chemicals, Mumbai, India.

Collection of Plant Ceropegia rollae
The plant Ceropegia rollae was collected from the Rajapur region of the Maharashtra India, identified and authenticated by Dr. Arun Chandore, Abasaheb Marathe College, Rajapur, Tehsil of Ratnagiri District, Maharashtra, India. The specimen was kept in the Abasaheb Marathe College, Rajapur ( Fig. 1-3).

Experimental
The plant material was dried in shade for 4 weeks, then it was dried in oven at 40 0 C for six hrs. Then dried plant material were crushed into fine powder. Both were kept in the glass bottles under inert atmosphere. Phytochemical testing carried out by given protocol and then other biological activity assays were carried using proper protocol.

Determination of total phenolic content
The total phenol present were determined by the known protocol. This protocol has the extract solution 0.3 ml with 1.5 ml of the reagent, 10% Folin-Ciocalteu's then 7.5 % of the sodium carbonate added. The mixture formed at dark for the 30 min. The absorbance was measured at 760 nm and can be done using the standard gallic acid curve.

Determination of antioxidant activity of the plant Ceropegia rollae
Antioxidant activity was inspected for the extract by using FRAP and DPPH assays. DPPH is well known standard. The FRAP reagent was prepared with 25 ml acetate buffer with 2.7 ml 2,4,6-tris (2-pyridyl)-Striazine (TPTZ) solution and 2.7 ml of the 20 mm of ferric chloride water solution. Each sample (150 µl)was mixed with 0.6 mg/ml methanol and 4.7 ml of the FRAP reagent and stirred for 5 min. The absorbance was measured at the 590 nm. The calibration curve method was used to study. The L ascorbic acid was used as standard (22).    water for 24 hrs in the gloomy condition. The absorbance was measured at 510 nm. The standard bend was linear at 27 to 800 µM of vitamin-C absorbance. All determination was carried using the complex of the DPPH radical by the sample, using the standard protocol (23).

% inhibition= [{Abs control -Abs sample}/Abs control] × 100.
Where Abs control is the absorbance of the DPPH radical with ethanol, Abs sample is the absorbance of DPPH radical with sample extract/standard.
We have studied DPPH activity at the various concentration as from the 0.05, 0.1, 0.2, and 0.3.

In-Vitro anti-inflammatory activity
For this, the albumin denaturation method was followed with modification. This reaction consists of the test extract with the 1 % aqueous solution bovine as albumin fraction. The pH of this reaction mixture was adjusted at 37 0 C. These are incubated at 37 0 C for 20 min, and heated above 50 0 C for 20 min. Then cooled, the turbidity formed in the solution was measured using the spectrophotometer at 660 nm. The percent inhibition of protein as denaturation was intended as follows:

% inhibition= [{Abs control-Abs sample}/Abs control] ×100
where Abs control is the absorbance of the DPPH radical with ethanol, Abs sample is the absorbance of DPPH radical with sample extract/standard (Table 2 and 3).

DPPH Scavenging Test
Quantitative measurement of radical scavenging property.

Determination of Anti-inflammatory Activity
Study of anti-inflammatory activity through in-vitro models were carried out on leaves and bark extracts by inhibition of the albumin denaturation. This was studied according to Muzushima and Kabayashi with slight modification at the doses of 200 mg/kg (Table 4 and 5), (9).

Phytochemical Analysis
The phytochemical analysis was carried for the leaves and bark extracts for saponins, terpenoids, flavonoids, tannins, and phenols using the fixed protocols. The FRAP and antioxidant activities of leaves and bark ethanol extracts were also studied ( Table 6).

Results and Discussion
The present study reports the phytochemistry, antioxidant and the anti-inflammatory activity of the leaves and bark of the plant Ceropegia rollae. The study revealed the presence of the saponins, terpenoids, flavonoids, tannin, phenolin the in leaves and stem of the plant, using the fixed protocols. Mostly the ethanolic extract and water extract were used for the antioxidant activity by the FRAP and the DPPH assays, and showed excellent activity compared with standard drug. Ethanol extract showed the good antioxidant activity than the water extract compared with the standard. The result of the anti-inflammatory study showed the inhibition of albumin denaturation by the bark and leaves of the plant. Ethanol extract of the leaves and stem showed excellent anti-inflammatory activity.

Conclusion
We have studied the new species, Ceropegia rollae for phytochemistry, anti-inflammatory and the antioxidant activity. This shows the presence of the various phytochemicals in the leaves and root of the plant. The leaves show the excellent antioxidant and anti-inflammatory activity than the bark. We can use these compounds as the natural antioxidant and antiinflammatory agent.