Comparative In vitro Anti Inflammatory Study on Alpha Amyrin Isolated from Bark Extracts of Ficus benghalensis. Linn and Alstonia boonei. De Wild

Introduction

India is a land of miraculous plant species which have been used traditionally for various kinds of ailments from the period of Vedas. Despite the emergence of modern allopathic system of medicine, a huge population still follows the indigenous systems like Ayurveda, Siddha, and Unani, which basically involves use of natural products, herbs and medicinal plants.¹ The presence of bioactive compounds with diverse chemical structures and potential pharmacological activities make plants marvellous candidates for cure of various diseases with an advantage of minimal side effects. Ancient literatures like Charaka Samhita and Sushrutha Samhita give an extensive description of various medicinal herbs. In modern medicine, plants play a vital role as raw materials for many important medicines. Allopathic medicines produce quick results in reducing ailments but these synthetic drugs are quite expensive and inaccessible for much of the population; in addition, they add up to the burden of serious adverse effects. This is where natural sources of medicines gain prominence as they are devoid of side effects, easily available and low in cost. The present work emphasises on the anti-inflammatory role of alpha amyrin. Inflammation is body’s defence mechanism against invading microbes or other harmful substances, chemicals or radiation which activates a vexatious and obnoxious complex constrain triggering the white blood cells by activating macrophages, leucocytes and mast cells resulting in rubor, calor, tumour and dolor or otherwise redness, heat, swelling or inflammation and pain. Inflammation results in liberation of precursors and mediators of inflammation such as arachidonic acid, prostaglandin, histamine, serotonin, cytokines, leukotrienes, interleukins, tumour necrosis factor, etc. This in turn elevates vascular permeability and facilitates migration of leukocyte infiltration to the site of inflammation.^2,3 Inflammation is associated with various levels of damage from minor cuts and wounds to major organ inflammation and damage. Inflammatory process is also involved in higher level systems such as brain where noxious chemicals, injury, infection, ageing, oxidative stress, wear and tear, triggers brain’s innate immune mechanism resulting in neuro inflammation which imparts irreversible damage to the brain and other parts of central nervous system. Microglia, innate immune cells of brain, etc. regulates and mediates the neuro inflammatory responses. Neurodegenerative diseases like Alzheimer’s, Parkinsonism, multiple sclerosis, etc. are all categorised under neuro inflammatory disorders.⁴

The Ficus benghalensis. Linn (Fb) is commonly known as Indian Banyan tree, belonging to the family Moraceae.⁵ It is an epiphyte and is otherwise known as ‘strangler figure’6 and it develops adventitious roots or the aerial prop roots as they mature. It has been referred as a treatment option for diarrhoea, haemorrhages, piles, vomiting, etc. in Ashtangahridaya and other ancient texts.⁷ It is used in treatment of leucorrhoea, osteomalacia, burning sensation, hemoptysis, hemorrhages, diarrhea, dysentery, diabetes, enuresis, ulcers, skin diseases, gonorrhea, leprosy. The latex is useful in neuralgia, rheumatism and lumbago bruises, mastitis, menorrhagia, ulitis, odontopathy, hemorrhoids, gonorrhea, inflammations, cracks of the soles and skin diseases.⁸ It has versatile bioactive component viz. β Sitosterol, esters, Quinic acid, α-Amyrin, Androstan17-one, Lupeolacetate, Diglycerol, glycosides, leukocyanidin, quercetin, sterols and friedelin, bergapten, flavonoid, galactose, inositol, leucopelar, rutin and tanins. ^9,10 It is also rich in ketones, polysaccharides, sitosterol and toglic acid.⁷ It also possesses antioxidant, anticancer, immunomodulatory, anticoagulant, antidiabetic, antiulcer, anti-inflammatory, anti-proliferative, hepatoprotective, anthelmintic, wound healing, antimicrobial, analgesic and antimutagenic activities.^11-13

On other hand, Alstonia boonei De Wild (Family: Apocyanaceae) (Ab) commonly known as ‘Scholar tree’, is also a good source of alpha amyrin. The bark is used in ethnomedicine for the management of malaria, ulcer, rheumatic pain, toothache, and inflammatory disorders, antimalarial, antipyretic, analgesic and anti-inflammatory properties, anthelmintic, diuretic, spasmolytic, hypotensive properties, immuno-stimulant property, antipsychotic and anxiolytic effect, antirheumatic, and antibiotic properties.^14-17 The main phytoconstituents include echitamidine, Nα-formylechitamidine, boonein, loganin, α-Amyrin, lupeol, ursolic acid, β-amyrin, alkaloids, cyanogenetic glycosides, flavonoids, terpenoids, steroids and saponins.18 Alpha amyrin, a pentacyclic triterpene isolated from Fb and Ab stem bark were studied and compared for their anti-inflammatory activities.

There are scanty reports on such comparative investigations of alpha amyrin isolated from bark of Fb and Ab for its anti-inflammatory activity through in vitro models. Such a study would help to identify a better source of alpha amyrin for its anti-inflammatory action. The study would be luminary to determine the possible anti-inflammatory activity of alpha amyrin isolated from the said plants and also worthwhile for the comparative study for further investigations.

Materials and Method

Collection of plant material

The bark of Fb and Ab were collected from Bengaluru, Karnataka. The plant was identified and authenticated by a plant taxonomist Dr. P. E Rajshekharan, IIHR, Bengaluru and the whole dried barks was preserved with the voucher specimen (KCP-PCOG/FB/330/2021- 22) and (KCP-PCOG/AB/331/2021-22) in Department of Pharmacognosy, Krupanidhi College of Pharmacy, Bengaluru, in the herbarium.

Preparation of plant crude extracts

The stem bark of Fb and Ab were collected and air dried at room temperature for two weeks and then coarsely powdered separately by hammer mill. Methanol was the solvent for extract preparation. Each 500 g of powdered drugs were used for the extraction using soxhlet method at temperature 40⁰ C for 4 hrs. The extracts were filtered separately through Whatman filter paper No 42. Further, they were concentrated to form thick, viscous crude form using rotary evaporator (Heidoph 4001 efficient), warmed on a water bath at temperature of 50°C to obtain semi solid crude extracts. Finally, the yield was calculated.

Chromatographic identification

The crude extracts were separately evaluated for the presence of alpha amyrin by Thin layer chromatography (TLC) followed by High performance liquid chromatography (HPLC) as per the literature reference.^19,20 After confirmation, isolation of the said compound was performed for both the extracts.

Isolation

Methanol extract 35 g was subjected to column chromatography using various solvents (from non-polar to polar solvents) and various fractions were collected. Each fraction was identified using TLC method and finally 100% ethyl acetate fraction identified the presence of desired compound i.e alpha amyrin. The same method was followed for both the extracts. Finally, at melting point, Liquid chromatography mass spectrometry (LCMS) and Fourier transform infrared (FTIR) studies confirmed the isolated compound as alpha amyrin.

In vitro anti-inflammatory activity

Inhibition of albumin denaturation

The determination of anti-inflammatory activity of alpha amyrin isolated from Fb and Ab stem bark was carried out as per standard method with slight modifications.²¹Inhibition of albumin denaturation was done according to the protocol. The reaction mixture consists of an equal volume of alpha amyrin of different concentrations (20– 100 μg/mL) and 1% aqueous solution of bovine albumin. The pH of the reaction mixture was adjusted using a small amount of 1N HCl. The samples of alpha amyrin were incubated at 37°C for 20 min and then heated to 51°C for 20 min. The absorbance was measured after cooling the samples at room temperature. The turbidity formed was measured at 660 nm using ultraviolet (UV)- visible spectrophotometer (Model: Shimadzu UV-1800) and aspirin (100 μg/mL) as standard. The percentage inhibition of protein denaturation was calculated using the following formula:

Anti-proteinase activity

The activity was performed by the standard protocol with some modifications.²² The reaction mixture (2 mL) containing 0.001% trypsin, 1 mL of 1mM Tris HCl buffer (pH 7.4) and 1 mL test sample of different concentrations (20-100 μg/mL) was incubated at 37°C for 5 min and then 1 mL of 0.02% (w/v) casein was added. The mixture was incubated for an additional 20 min at 37°C. 2 mL of 2% perchloric acid was added to the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank and aspirin (100 μg/mL) as standard. The percentage inhibition of proteinase inhibitory activity was calculated using the following formula:

Anti-lipoxygenase activity

Anti-lipoxygenase activity was studied with minor modifications, using linoleic acid as substrate and lipoxidase as an enzyme.²³ Test samples were dissolved in 2M borate buffer pH 9.0 (0.25 mL) and 0.25 mL lipoxidase enzyme solution (20,000 U/mL). The reaction mixture was incubated for 5 min at 25°C. Then, 0.6 mM linoleic acid solution (1.0 mL) was added. The reaction mixture was vortexed, then 1 mL test sample of different concentrations (20-100 μg/mL) was added. The absorbance was measured at 234 nm. Aspirin was used as a reference standard (100 μg/mL). The percent inhibition was calculated by the following equation:

Statistical analysis

Mean and SEM (standard error of the mean) were calculated for all the parameters in the experiment. Comparison of means was done using the one-way ANOVA to determine the significant difference at p < 0.05. 

Results

Yield

The yield of the crude methanol extracts was separately calculated and result is depicted in Figure 1. The yield was 43.12 g and 34.48 g for Fb and Ab bark extracts, respectively.

TLC identification

TLC separation was performed on silica gel 60 F254 as stationary phase for both the extracts. Dichloromethane and toluene with ratio of 9.5:0.5 (v/v) was used as mobile phase. Detection and quantification were performed by densitometry at 586 nm. The Rf of the sample and standard was found to be 0.46 and 0.49 for Fb and Ab extracts, respectively (Figure 2).

HPLC study

Further, HPLC study for the extracts and standard was performed at 206 nm with the mobile phase acetonitrile and water in the ratio of 65:35. The flow rate 1.0 mL/ min was maintained. Injection volume was 10 µL. The result revealed that standard alpha amyrin showed peak at Rt 16.1 min whereas the same of Fb and Ab extracts showed the peak at 16.1 min which was similar with the standard (Figure 3, 4a, 4b).

Isolation of constituent

Column chromatography of the extracts (35 g of each extract) was performed and alpha amyrin was isolated in 100% ethyl acetate fraction which was further purified and a white mass precipitate was obtained. The amount was 0.42 mg and 0.23 mg for Fb and Ab bark extracts, respectively. At first, melting point was performed using Fisher-Johns apparatus which resulted 185.8^o C. Thereafter, FT-RI and LCMS studies were performed and result is depicted in figure 5 and 6 respectively.

IR study resulted in some important chemical bonding of the constituents and these are mentioned in Table 1.

In vitro anti-inflammatory activity

Inhibition of albumin denaturation

In the present study, the inhibition of protein (albumin) denaturation was analysed and was found that alpha amyrin isolated from methanolic extract of Fb bark exhibited superior in vitro anti-inflammatory activity compared to alpha amyrin isolated from methanolic extracts of Fb and Ab, with former showing 88.03±0.63% at 100 µg/mL whereas the latter exhibited a value of 88.34 ± 0.46 % at 100 µg/mL when compared with standard aspirin (100 %) at 100 µg/mL concentration. Results are represented in figure 7.

Anti-proteinase activity

The evaluation of proteinase inhibitory action of alpha amyrin from methanolic bark extracts of Fb and Ab was carried out and both showed significant proteinase inhibitory action with maximum inhibitory action exhibited by alpha amyrin isolated from Fb bark with a result of 98.67%, and a value of 98.13% at 100 µg/mL concentration was observed with the same alpha amyrin isolated from methanolic extract of Ab (Table 2).

All values are expressed as mean±SEM (n=3), statistically significant at (*) =p<0.05 (**)=p<0.01; Values are compared with standard by using one way ANOVA followed by Dunnette’s multiple comparison test.

Anti-lipoxygenase activity

A similar trend was followed for the anti-lipoxygenase activity and was found to be maximum with alpha amyrin from methanolic bark extract of Fb with 96.84 %. On the other hand, a slightly lesser result was observed with alpha amyrin from methanolic extract of Ab by inhibition of 96.19% at 100 µg/mL concentration (Table 3).

All values are expressed as mean ± SEM (n=3), statistically significant at (*) = p<0.05 (**)= p<0.01; Values are compared with standard by using one way ANOVA followed by Dunnette’s multiple comparison test.

Discussion

The stem barks of Fb and Ab were subjected to methanolic extraction and the yield was estimated separately. Solvent plays an vital role in availing maximum yield from any plant specimen as the separation of phytocompounds occurs based on the polarity. Before experimentation, various solvents were used for the extraction, and methanol was identified as the best choice of solvent.²⁴ Based on this concept, in the present study, methanol was used as the solvent for bulk extraction. The methanolic extract of stem bark of Fb gave a higher yield of alpha amyrin than Ab extract and the reason could be because of the presence of high content of bioactive constituents. Previous literature also revealed similar reason for the higher yield of plant extract.²⁵ Fb and Ab are good sources of alpha amyrin and the present study confirmed that both the extracts yield alpha amyrin. TLC and HPLC were performed for the extracts and both of them demonstrated the presence of alpha amyrin. These results are similar to the earlier reports.^26,27

Column chromatography technique was used for the isolation of the desired bioactive compound and ethyl acetate as solvent served the purpose. The experiment was performed similar to the earlier scientific report.²⁸

The yield of isolated compound was higher in Fb compared to Ab bark extract. The isolated constituent was confirmed as alpha amyrin by testing its melting point, and subjecting to LC-MS, FT-IR studies, which also correlated with the previous scientific reports.²⁹ The present experiment was performed to identify any significant differences in the anti-inflammatory activity of alpha amyrin isolated from Fb and Ab extracts, but non-significant results were obtained. The study indicated that any activity of the isolated compound was independent of any parameter with respect to different sources, but the study revealed that Fb was a better source for isolation of the bioactive compound alpha amyrin, compared to Ab bark source. 

Conclusion

The present study showed that methanol is the best solvent that yielded maximum extract and that Fb stem bark is the better source for isolating alpha amyrin compared to Ab stem bark. The desired bioactive compound alpha amyrin was identified with TLC and HPLC studies. Furthermore, the isolated compound was confirmed by testing its melting point and through FT-IR and LCMS studies. Using alpha amyrin isolated from two different sources, in vitro anti-inflammatory study was performed. The study showed that alpha amyrin effectively inhibited enzyme activities in a concentration dependent manner, but the study indicated that a non-significant variation exists for the anti-inflammatory activity of alpha amyrin isolated from the two different sources. Altogether the study suggests that Fb extract is a superior choice over Ab extract for the isolation of alpha amyrin because of the higher yield of isolated constituent and overall antiinflammatory activity.

Conflict of Interest

Authors declared no conflict of interest.