Ophiorrhiza mungos var . angustifolia – Estimation of camptothecin and pharmacological screening

Ophiorrhiza mungose var. angustifolia (Thwaites) Hook. f (FamilyRubiaceae) is a recently identified plant from Ophiorrhiza species in Western Ghats of Kerala. The plant is a promising candidate for the production of camptothecin (CPT) a high value anticancer compound. Preliminary screening of hexane and methanol extract revealed the presence of phenolics, flavonoids, caumarins, steroids, terpeanoids, saponins, carbohydrates and alkaloids. Camptothecin was estimated from methanol extract using high performance liquid chromatography and the level of CPT was 297.94 ± 2.27 μg/g dry weight. The in vitro antioxidant assay revealed both extract showed moderate level of total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric chloride reducing power assay, phospho-molybdate assay of total anti-oxidant capacity and nitric oxide scavenging activity assay. Antimicrobial study reveals that only hexane extract inhibits pathogenic bacteria and fungus. Overall these findings will lead to isolation of active compounds other than camptothecin, elucidate them against wider range of bioactivity studies to find new therapeutic principles.


Introduction
Ophiorrhiza belongs to the dicotyledonous family Rubiaceae and is represented by 150 species, distributed mainly in the Indo-Malay region (1).Southern western Ghats of India is considered as a rich repository of Ophiorrhiza as evidenced by the survey reports showed, 21 species and 2 varieties, of which 13 species are endemic (2,3,4,5).Other important phytochemicals, including derivative of CPT, pumiloside, luteolin, harman, tetrahydro alastonine, bracteatine, blumeanine, strictosidinic acid, lyalosidic acid are also present in varying quantities in certain species of Ophiorrhiza (6).
Camptothecin (CPT), a monoterpnene indole alkaloid and is regarded as one of the potent inhibitor of topoisomerise 1. CPT and its derivatives are also reported to be very active anticancer agents against colorectal and ovarian cancer (7,8).CPT originally isolated from Camptotheca acuminata (9) is also reported in Nothapodytes foetida (10), Tabernaemontana heyneana (11) and various Ophiorrhiza spp.(12).
O. mungos var.angustifolia is distributed all over peninsular India and Sri lanka (13), and is commonly called Indian snake root or mangoose plant.The plant is reported to have second highest CPT content among different Ophiorrhiza species in the southern Western Ghats (12).
Recent reports reveals, owing to higher level of camptothecin in O. mungos var.angustifolia researchers develop an efficient in vitro regeneration protocol for the propagation and production of CPT from this species (14).Phytochemicals other than camptothecin such as phenolics, favanoids, coumarins, steroids, terpaoids and alkaloids are reported from in O. mungos and O. prostrata where they act as antioxidants and free radical scavengers (15).So far no extensive in vitro antioxidant and antimicrobial screening have been reported on O. mungos var.angustifolia.The main objective of the present study to carry out the qualitative and quantitative evaluation of camptothecin, estimation of total phenolic compounds and evaluation of in vitro antioxidant activity and antimicrobial activity of O. mungos var.angustifolia.

Materials and methods
The whole plant of O. mungos var.angustifolia (Fig. 1A) was collected in June 2017 from Idukki -Munnar -Neriyamangalam (Latitude 10.061774° N; Longitude 76.760652° E).The whole plant was identified and authenticated the authors.A voucher specimen (No.TBGT79923) has been deposited at Jawaharlal Nehru Tropical Botanic Garden and Research Institute, India.

Preparation of plant extract
The dried and ground plant material (20 g) was successively extracted with hexane and methanol 5 h each.The extracts were concentrated to dryness under reduced pressure using rotaevaporator (Heidolf, Germany).The completely dried extract was used for further analysis.(16).

Qualitative assay of camptothecin -Thin layer chromatography (TLC)
The active phytochemical camptothecin in O. mungos var.angustifolia was detected and estimated using TLC.20 µL of standard camptothecin (mg/mL in methanol) and methanol extracts of plant were loaded on a precoated TLC plate (5 x 10 cm, Silica gel 60 F254) (Merck, Germany) and chromatographed in the solvent systems toluene: acetonitrile: glacial acetic acid (65:35:1 v/v/v) (14).The co-chromatographed authentic sample of CPT was used to detect the presence of CPT in the sample lane on the TLC plate under the UV chamber and Rf value was calculated (17).

Quantitavie assay -HPLC Estimation
The methanol extract of O. mungos var.angustifolia was analysed using HPLC Shimadzu Japan).The system attached a UV/VIS Detector (Prominance SPD M20 A diode array detector).Data acquisition and instrumental control were performed using Shimadzu Lab solution version 5.73.Separation of the compounds was performed on a general purpose Shimadzu C-18 column (250 x 4.6 mm, 5 µm particle size, 5 µm) and the isocratic mobile phase consisted of 100% methanol (Spectrochem, India -HPLC grade).The flow rate was 1.0 ml/min and the injection volume was 10 µL.Pump consists of (LC 6 AD) system interface (CBM-20A) and a high pressure adjustable volume dynamic mixer.The analysis was performed at room temperature (25ºC) and the compound was detected at 254 nm. 1 -500 ppm of authentic sample was run in HPLC column for calibration curve.Plant extract filtered using a 0.22 μm filter (Millipore) inject separately.At the same retention time of standard the peak area of the extracts were measured and quantified using the equation from the calibration curve, f(x) =21648.5*x+8030.52.The results are expressed in mean ± standard deviation (18).

In vitro Pharmacological screening of O. mungos var. angustifolia
Estimation of phenolics using Folin Ciocalteu method: Total phenolic contents of each extract were determined using a modified Folin-Ciocalteu colorimetric method (19).Folin ciocalteu method is a simple procedure to estimate the total phenolics and thus total reducing power of the extracts.Different concentrations (10 -100 mg/mL) of the extract were transferred to reaction tubes and were made up to 0.5 mL.To this 3mL of water was added followed by Folin Ciocalteu Phenol reagent.Immediately 0.75 mL of 20% (w/v) sodium carbonated was added, followed by 0.95 mL of water.The reactants were mixed and were incubated for 30 min at 30ºC.The absorbance was measured at 765 nm using a UV-visible spectrometer.A standard curve method was adopted for the estimation, using 5 different concentrations of the extracts.Gallic acid (1 mg/mL) served as the positive control; the reducing power was expressed as gallic acid equivalent (GAE).

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay:
The antioxidant activity of the extract was determined by the 1,1-diphenyl-2picryl-hydrazyl (DPPH) assay, as described earlier with some modifications (20).Briefly 100 -500 µg/ mL of the plant extract were taken in 5 mL screw cap tubes and were made up to 1 mL with methanol.1 mL of methanol devoid of extracts served as the control.To these tubes 1 mL of 0.2 M of DPPH solution in methanol was added and incubated for 1 h at room temperature.Ascorbic acid served as a comparative control.The absorbance was measured at 517 nm in a UV-Visible spectrometer (UV2100, Shimadzu Corporation, Japan).Ascorbic acid is used as the positive control.The anti-oxidant activity was measured as 50 % radical scavenging activity (IC50 value) by the formula: % Inhibition = OD control -OD of test x 100

OD of control
Ferric chloride reducing power assay: Various concentrations of the samples were taken and were made up to 0.5 mL with 0.2 M phosphate buffer (pH 6.6).To this 1 mL of 1 % (w/v) potassium ferric cyanide is added and shaken well incubated at 50°C for 20 min in a water bath.At the end of the 20 min of 10% TCA was added and mixed.Then 1.5 mL of water was added to this mix followed by addition 0.3 mL of ferric chloride.The colour developed was measured spectrometrically.Ascorbic acid was used a comparative control (21).

Phospho-molybdate assay of total anti-oxidant capacity:
The extracts were taken at desired concentrations and were made up to 200 µL with water.To these tubes 2 mL of phosphomolybdate reagent is added and was incubated at 90°C for 90 min.The increase in the green colour in relation to the antioxidant content of the extract is evaluated by the increase in absorbance of the test sample.
The absorbance was measured at 695 nm in UV-Visible spectrophotometer (Shimadzu, Japan).A standard curve method was adopted for the estimation, using 5 different concentration of the extracts.Gallic acid was used as positive control (22).

Nitric oxide scavenging activity assay:
The nitric oxide scavengers in the extracts mop measured spectrometrically.Various concentrations of the extracts were taken in the reaction tube and were made up to 0.5 mL with 0.01 M PBS (pH 7.4).To this 10 mM of sodium nitroprusside (SNP) dissolved in 0.01 M PBS (pH 7.4) was added and mixed.The tubes were then incubated for 2 h at 30°C.Post incubation, 0.5 mL of Greiss's reagent was added to the samples and mixed and incubated for 20 min.The colour developed was spectrometrically measured at 546 nm in a UV-Visible spectrophotometer (Shimadzu, Japan).A standard curve method was adopted for the estimation, using 5 different concentrations of each of the extracts (23).

Antimicrobial assay:
The agar well diffusion method was performed to screen for antimicrobial activity of hexane and methanol extracts of O. mungos var.angustifolia (24).The gram positive bacteria (Escherichia coli, Salmonella typhi and Klebsiella pneumoniae), gram negative bacteria (Stephylococcus aureus and Bacillus subtilis) and fungal strains (Aspergillus niger and Colletotrichum) were used for the present study.The both extracts were taken as mg/mL in DMSO and streptomycin was prepared in sterile distilled water as positive control and negative control was pure DMSO.Nutrient medium (Merck) for bacteria and Saboured Dextrose media (SDA) (Himedia, Bangalore) for fungus was prepared according the manufactures instruction.
Nutrient and SD broth were prepared in test tubes and plugged with non-adsorbent cotton; bacterial and fungal cultures were inoculated in the appropriate medium from the stock and kept in rotor evaporator for overnight at 100 rpm.Nutrient agar and SD agar medium were immediately after autoclaving, it was cooled and 20 mL each dispensed into glass petri dishes to give a uniform depth of approximately 4 ± 0.5 mm.The 100 µL of turbid bacterial and fungal suspension were inoculated and swabbed.Wells of 6mm size were made using sterile cork borer and the rim of the agar is swabbed.Plant extracts and controls were then dispensed (50 µL) into the wells, the plates are then sealed with para film and incubated at 37°C for 18-24 h.After 18-24 h of incubation, each plate is examined.The diameter of the zones of compete inhibition were recorded in the test as well as the control well.Assay was done three times and average of inhibition zone with standard deviation was calculated.

Statistical Analysis
Statistical analysis was carried out with IBM SPSS statistics ver.20, and results are expressed as means ± standard deviation.

Extraction yield
The yield of extraction depends on the solvent with varying polarity, temperature, extraction time, and composition of the sample.In this work, O. mungos var.angustifolia, hexane and methanol extracts gave 6.75 % and 15.58 % respectively by soxhlet method.In the present study extreme low and high polar solvents were selected where as the camptothecin is a polar compound and can be extracted with methanol or chloroform (12,17) while the previous report states that methanol is more suitable solvent for camptothecin isolation (12,15).This shows that the extraction yield increases with increasing polarity of the solvent used in extraction.The antioxidant and antimicrobial activity also tested the same solvent extracts.The results of this study are in agreement with the extraction yields of rice bran (25) and some medicinal plants (26).

Preliminary phytochemical screening
The phytochemical investigation of the hexane and methanol extracts of O. mungos var.angustifolia revealed that phenolics, coumarins, flavonoids, steroids, terpenoids, saponins carbohydrate and alkaloid were present in the extract.In the present study qualitative evaluation of the chemical constituents of methanol extract of O. mungos var.angustifolia showed the presence of various classes of compounds including, phenolics, saponins, terpenes, steroids, carbohydrates and alkaloids.The presence of these classes of compounds contributes different biological activities.This might be attributed to their free radical scavenging, anti-microbial and anticancer activities (27,28).

Quantitative assay of camptothecin -HPLC Estimation
Estimation of camptothecin is measured using Standard graph method in HPLC is 297.94 ± 2.27 µg/g dr.wt.(Fig. 2).The retention time of camptothecin is 4.184 min.Several approaches have been employed for the analysis of CPT, mainly high performance liquid chromatography (HPLC) (32).Recently, alternative simple and sensitive methods involving HPLC coupled with mass selective detection have been improved and these have been shown to be more accurate than conventional HPLC system.

Total phenolic content
The total phenolic content of the hexane extract, calculated from the calibration curve (R2 = 0.998), was 27.28 ± 0.579 gallic acid equivalents/g, and methanol extract (R2 = 0.999) was 60.67 ± 0.760 gallic acid equivalents/g (Fig. 3; Table 1).Phenolic compounds have redox properties, which allow them to act as antioxidants (33).The free radical scavenging ability of plant extract helped by their hydroxyl groups, the total phenolic concentration could be used as a basis for rapid screening of antioxidant activity.As this is the first report on the antioxidant activity of O. mungos var.angustifolia through phytochemical analyses should be done to identify the active phenolic components from both extracts.

In vitro anti oxidant assay
Antioxidant potential of O. mungos var.angustifolia was determined by using DPPH, Ferric chloride reducing power assay, Phosphomolybdate assay of total anti-oxidant capacity and Nitric oxide scavenging activity assay.The IC50 value was calculated and mentioned in the Table 1.
Plants rich in secondary metabolites have antioxidant activity due to their redox properties and chemical structures.The hexane and methanol extract of O. mungos var.angustifolia had moderate antioxidant activity against investigated free radicals.The DPPH radical is widely used in assessing free radical scavenging activity because of the ease of the reaction.IC50 value of DPPH scavenging activity was 65.37 ± 4.97 µg/mL in hexane and in methanol extracts while that of the control, ascorbic acid, was 66.69 ± 5.63 µg/mL.The molecule DPPH is characterized as a stable free radical by virtue of the delocalisation of the spare electron over the molecule as a whole, so that the molecule does not dimerize, as would be the case with most other free radicals (34).
In ferric chloride reducing power assay, the Fe 3+ -Fe 2+ transformation in the presence of the extracts was assessed.The reducing power of extracts and ascorbic acid, used as reference compound, were assayed and the results are shown in Table 1.Both extracts have moderate level of reducing power activity than ascorbic acid.In our work a correlation between reducing power activity of extracts and polyphenol and flavone content of them can be observed as in other studies (35,36).
The present study hexane (IC50 = 105.2± 5.00 µg/mL) and methanol (IC50 = 328.7 ± 7.67 µg/mL) extracts of O. mungos var.angustifolia may be act as antioxidant by inhibiting the production of nitric oxide.Nitric oxide is a lipophilic molecule.At physiological pH it reacts with oxygen to produce stable products (nitrate and nitrite), the quantities of which can be determined using Griess reagent (37).Scavengers of nitric oxide compete with oxygen and inhibit the production of nitric oxide (38).
Total antioxidant capacity of hexane and methanolic extracts was determined using phosphomolybdate assay (Table 1).The assay is based on the fact that molybdenum (VI) is reduced to molybdenum (V) in the presence of a reducing agent (antioxidant), forming a green phosphomolybdate (V) complex, which can be evaluated spectrophotometrically at 765 nm (39).The assay involves an electron transfer mechanism.Many natural products, including phenols and flavonoids, can cause this reduction.

Anti-bacterial and antifungal studies
The hexane extract of the plant showed activity against gram positive bacteria; Bacillus subtilis and gram negative bacteria; E. coli, Klebsiella pneumonia, Staphylococcus aureus.It also showed moderate activity against the fungi Colletotrichum gloeosporioides and Aspergillus niger (Supplementary data 1).The antibacterial activity of the hexane and methanol extract shown in the Table 2.
Plants with their bioactive chemical compounds, serve as plant defense mechanisms against invasion by microorganisms (40,41).It was due to the differences in the phyto-constituents of the extracts.In the present analysis it was observed that the plant extracts have considerable bactericidal activity against gram positive, gram negative and fungus in hexane extract only while there is no antimicrobial activity in methanol extract and camptothecin (Suppementary data 1).This may be due the methanol extract has only camptothecin and its derivatives, HPLC data corroborate with the result.O. mungos var.angustifolia is herbaceous medicinal plant and its large scale multiplication is a good alternative system for the isolation of camptothecin in industrial level.

Fig. 3 .
Fig. 3.Text figure is the standard graph of gallic acid for estimating total phenolic content of O. mungos var.angustifolia.The total phenolic content (x) was calculated from the regression equation of calibration curve where 'y' is the absorbance.Data represent the mean of three replicates ± SD.

Table 1 .
Antioxidant effect (IC50 value, µg/mL) on DPPH radicals, Ferric chloride reducing power assay, Nitric oxide and Phospho-molybdate assay for hexane and methanol extract of O. mungos var.angustifolia.Standard for DPPH, Ferric chloride -ascorbic acid; Phsphomolibdate, Nitric oxide -Gallic acid.Each value in the Table is represented as mean ± SD (n = 3).Means not sharing the same letter are significantly different (LSD) at p < 0.01 probability level in each column. *

Table 2 .
Antibacterial and antifungal activity of hexane and methanol extract of O. mungos var.angustifolia