Isolation and characterisation of cryptolepine from SidaacutaBurm and its antimycobacterial activity Krishnaveni A akrishnaveni72@rediffmail.com Asst.Prof, 1 II M. Pharm, Department of Pharmacognosy, College of Pharmacy, Madurai Medical College, Madurai, Tamilnadu, India. India 10 08 2020 10 08 2020

IJAMSCR |Volume 6 | Issue 2 | Apr - Jun - 201 8

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Research article Medical research

Isolation and characterisation of cryptolepine from <italic>Sidaacuta</italic>Burm and its antimycobacterial activity Krishnaveni A*<sup>1</sup>, Ezhilarasan B<sup>1</sup>, Iyappan<sup>1</sup> A, Abdul HasanSathali A<sup>2</sup>

* 1 Asst.Prof, 1 II M. Pharm, Department of Pharmacognosy , College of Pharmacy, Madurai Medical College, Madurai, Tamilnadu , India.

2 Principal, College of Pharmacy, Madurai Medical College, Madurai, Tamilnadu , India.

*Corresponding Author: Krishnaveni A

Email id: akrishnaveni72@rediffmail.com

ABSTRACT Background

Sidaacutais a shrub indigenous to pantropical areas, widely distributed in regions and found in pastures, waste lands, cultivated lands, roadsides, lawns and planted forests. Sidaacuta is ethanomedically used as treatment of diuretic, asthma, fever, headache, cough, cold, ulcer, anthelmintic, snake bite, urinary disease, female disorders, sedative, eczema, kidney stone, elephantiasis, testicular swelling, poultice for dandruff, rheumatic affections, facial paralysis, pulmonary tuberculosis and gonorrheae.

 Methods

Hydro-alcoholic extract of Sidaacuta was chromatographed (Column) to isolate cryptolepine. The isolatetd SA-I was subjected to various spectral studies such as ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy and mass spectroscopy. The hydro-alcoholic extract of Sidaacuta and isolated SA-I were evaluated for its anti-mycobacterial studies by Luciferase reporter phage assay method using mycobacterial tuberculosis standard strain of H37RV.

 Results

The isolated compound (SA-I) showed the spectral data of cryptolepine. The percentage inhibition of hydro alcoholic extract of Sidaacuta (500 & 1000µg/ml) against mycobacterium tuberculosis showed antimycobacterial effect was found to be 99% in comparison with isoniazid (1ug/ml) 92% and rifampicin (1µg/ml) 96%. Whereas isolated cryptolepine did not any sensitiveness towards mycobacterium tuberculosis.This research indicatecryptolepine did not show any antimycobacterial effect. It specifyantimycobacterial effect of hydro-alcoholic extract of Sidaacuta may be due to the combined nature of cryptolepine with other phytoconstituents.

 Conclusion

The present research draws the conclusion that this Sidaacutaplant showed extreme antimycobacterial effect, which may be due to the phytoconstituents.

Keywords: Antimycobaterial, Cryptolepine, Sidaacuta,Malvaceae

 INTRODUCTION

Sidaacuta is shrub indigenous to pantropical areas, weed is frequently found in pastures, waste lands, cultivated lands, roadsides, lawns and planted forests.

Sidaacuta used in ayurvedic preparation as diuretic, sedative, abortifacient for the treatment of, asthma, fever, headache, cough, cold, ulcer, anthelmintic, snake bite, urinary disease, female disorders (Mudaliar, 1998) [19] eczema, kidney stone, elephantiasis, testicular swelling, poultice for dandruff, rheumatic affections, azoospermia, oligospermia, spermatorrhea, leucorrhoea, wounds, sciatica, nervous and heart disease, facial paralysis, pulmonary tuberculosis, gonorrheae (Ananil, K, et al 2000, [4]Silja, VP et al.,2008, Oliver Teneet al.,2017, Saraswathyet al.,1998)

The literature survey of the plant revealed the presence of tannin, saponin, flavonoid, terpenoids, cardio glycoside, vitamin composition was thiamine, niacin, ascorbic acid, tocopherol, riboflavin and mineral composition was calcium, magnesium , zinc, steroids (ecdysterone, β-sistosterol, ampesterol), phenolic compounds ( evofolin-A and B, scopoletin, loliolid and 4-ketopinoresinol, polyphenol, sesquiterpene, alkaloid cryptolepine, quindoline and quindolinone and fixed oil (Nwankpa, P et al.,2015 [20], Konate, K et al., 2010 [15], Jang, DS et al.,2003, [15]Palaksha, MN et al 2012) [27, 28]

The plant exhibited various pharmacological activities such as antibacterial (DamintotiKarouet al 2005), antimicrobial(Sanganuwanet al.,2006) larvicidal and reppellent (MarimuthuGovindarajanet al., 2010), gastric anti-ulcer (Akilandeswariet al., 2010), insecticidal (Adeniyi, SA et a.,2010) [1], hypoglycemic (Okwuosa, CN. et al.,2011) [22], anti-pyretic (Sharma, R.et al.,2012) [35], anthelmintic (Palaksha, MN. et al., 2012) [29], antioxidant and thrombolytic (EntazBaharet al.,2013) [8] electrolytes and organ function parameters (Enemor, VHA. et al., 2013) [9], diuretic and anti-urolithiatic (Palaksha, MN et al., 2015) [29], invitro stability and aggregatory (ObiomaBenedethEze, et al.,2016) [23, 24] anti –inflammatory (ObiomaBenedethEze. et al., 2016) [24, 25] alpha amylase Inhibitory (KemiFeyisayoet al.,2017), hepataprotective(Sridevi.et al.2009) calcium oxalate crystal growth inhibitory (Vimala,Tet al., 2012) [38] corrosion inhibitory (Umoren., et al.,2016) [37], antiplasmodial (Benzouzi. et al., 2004) [5], analgesis (Oboh, IE.et al., 2005) [26], anti-venom (Otero.et al.,2000) [26], anti-malaria (DamintotiKarouet al., 2003) [6] anti-ulcer (Malairajan, P et al., 2006), wound healing (Akilandeswari, S et al.,2010) [2, 3], cytototoxicity (Pieme, et al., 2010) cardiovascular (Kannan, RR et al., 2012), antifungal (Jindal Alka., et al 2012) [12], anticancer (Mahesh Thondawada. et al., 2016) [18]

The modern medicine causes hepatitis, lethargy, rashes, fever, acne, arthralgia, headache, redness and bone pain.

An attempt was taken to investigate isolated SA-I and invitroantimycobacterial effect for this plant.

 MATERIALS AND METHODS Plant collection & authentication

Fresh leaf of SidaacutaBurm were collected from the komanampatty village Dindigul (Dist), (Tamil Nadu) during the month of August-2017 was authenticated by DR. D.Stephen, M.Sc., Ph.D., Assistant Professor, Department of Botany, American College, Madurai-20.The herbarium of this specimen was kept in the department for further references

 Preparation of hydro-alcoholic extract of <italic>Sidaacuta</italic>Burm<italic>. </italic>(HAESA) Procedure

The shade dried and coarsely powdered leaf of SidaacutaBurm. (Leaf) was defatted with petroleum ether (60-80˚c). The residue was dried and extracted with hydro-alcohol (70%) by maceration until the complete extract of the material and filtered. The extract was concentrated under reduced pressure to obtain a solid residue (dark brown).

 Isolation of compound SA-I

Powdered, oven-dried (600 C) leaves of Sidaacuta 50gm were defatted with 100 ml of petroleum ether for 8 hours. The marc was extracted by soxhlet with methanol 100 ml Methanol for 8 hours the extract was evaporated under reduced pressure to give a residue 3.4gm which was thoroughly mixed with AcOH (5% 500ml). The mixture diluted with water (250ml) and allowed to stand overnight. The mixture was filtered affording a clear solution and some insoluble material.The aqueous filtrate was made basic with aqueous ammonia to PH 9.5 and extracted with CHCl3 (500ml). Extracts were dried over anhydrous MgSo4, filtered and evaporated to afford a dark alkaloidal residue (450mg). The alkaloidal residue was dissolved in CHCl3 (25ml) Adsorbed on to Al2O3 (20gm) and chromotographed over Al2O3 (200gm). Elution was conducted successively with petroleum ether 40-60 (500ml) followed by petroleum ether-CHCl3 (500ml) and CHCl3 (500ml) CHCl3- MeOH (9:1) 500ml and finally MeOH (500ml). QUINDOLINE was obtained as yellow solid after column chromatography and preparative tlc on AlO3 in chloroform. Which gave an orange colouration with Dragendorff’s reagent, which is evidence for quindoline alkaloids. Elution of the column with MeOH (9:1) afforded a violet residue.it crystallized from aqueous ethanol as long violet needles shaped named as SA-I

Spectral analysis of the Isolated SA-I subjected to ultraviolet spectroscopy infrared spectroscopy, nuclear magnatic spectroscopy, mass spectroscopy and the results are displayed in Figures:1,2,3,4 and 5.Tabulated in: Tables: 1,2,3,4 and 5

 DETERMINATION OF ANTIMYCOBACTERIAL ACTIVITY OF HAESA Luciferase reporter phage assay (LRP)

Antimycobacterial effect was determined forhydro-alcoholic extract of SidaacutaBurm as per (Papitha.net al 2013)

 Procedure

Standard strain H37RV and a clinical isolate of M.tuberculosisresistant to S, H, R& E were grown in Middlebrook 7H9 complete medium 12 with and without extracts of SidaacutaBurm. for 3 days at 37°C. Luciferase Reporter Phage Assay10 was done using concentrations of 500 and 1000 μg/ml of SidaacutaBurm extracts and isolated SA-I. Fifty-microliter bacterial suspension equivalent to MacFarlands No.2 standard was added to 400 μl of G7H9 with and without the test compound. For each sample, two drug-free controls and two drug concentrations were prepared and this set up was incubated for 72 h at 37°C. After incubation, 50 μl of the high titer Luciferase reporter phage (phAE129) and 40 μl of 0.1 M CaCl2 were added to all the vials and this setup was incubated at 37°C for 4 h. After incubation, 100 μl of the mixture was taken from each tube into a luminometer cuvette and an equal amount of working D-luciferin (0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was added. The RLU was measured after 10s of integration in the Luminometer. Duplicate readings were recorded for each sample and the mean was calculated. The percentage reduction in the RLU was calculated for each test sample and compared with control. The experiment was repeated when the mean RLU of the control was less than 1000. The results are tabulated in Table: 6 and displayed Figure: 6

 RESULTS AND DISCUSSION

Figure :1 UV spectrum of SA-I

The methnolic(100%) fraction of HAESA was pooled and the named as SA-I.The isolated compound SA-I was dissolved in methanol and was scanned under UV range from 200-600 nm in the UV-Visibble spectrophotometer. The isolated compound SA-I showed absorbance maxima at 255nm, 274nm and 331,which indicates the presence of an aromatic ring.

Figure: 2 Infrared spectroscopy of isolated SA-I

Table: 2 Interpretation of Infrared spectrum of SA-I

S.no Standard IR Range (cm -1 ) Observed IR range(cm -1 ) Intensity Types of band Functional group
1 3200-3600 3300 Strong, Broad Stretching OH, Alcohol
2 2850-300 2904, 2980 C-H-, Alkane Strong Stretching C-H-, Alkane
3 2100-2270 2264, 2231 Variable not present in symmetrical alkynes Stretching -C≡C-, Alkyne
4 1620-1680 1645 Variable Stretching -C‗C, Alkane
5 1350-1480 1452, 1419 Variable bending -C-H, Alkane
6 1345-1385 1382 Strong two bonds Stretching N-O, Nitro
7 1080-1360 1325, 1274,1085 Medium Stretching C-N, Amine
8 1000-1300 1043 Strong Stretching C-O, Ether
9 675-1000 875 Strong Stretching ‗C-H, Alkane

The isolated compound SA-I was scanned under IR range from 600cm-1- 4500cm-1 in the IR spectrophotometer. The IR spectrum of isolated SA-I shows a broad absorption band at 3300 cm-1 indicates (alcoholic –OH), 2904cm-1 and 2980cm-1 (alkane-C-H), 2264 cm-1 and 2231cm-1 (Alkyne-C≡C) 1645cm-1 (Alkane C-C,) 1452cm-1 and1419cm-1 (-C-H alkane), 1382cm-1 (nitro N-O), 1325cm-1,1274cm-1 and1085cm-1 (Amine-C-N ),1043cm-1 (Ether-C-O), 875cm-1 (Alkane-C-H),which indicates unsaturated compound with tertiary amine linkage.

Figure: 3 1 H NMR spectrum of SA-I

Interpretation of <sup>1</sup>H NMR spectrum of SA-I

The 1H NMR of isolated SA-I showed an intense singlet at δ 5.83ppm with integral of 36mm corresponding to 3H of the methyl group of SA-I at position 5.Since the methyl group at position 5 of SA-I has no neighbouring protons to couple it. It gave a singlet peak at 5.83ppm typical of signal produced by methyl group attached to tertiary nitrogen.

The 1H NMR of isolated SA-I showed aromatic multiples from δ 7.467 to 8.808 ppm aromatic singlet at δ 9.50 ppm corresponding to eight aromatic proton and isolated aromatic hydrogen of SA-I respectively. The aromatic singlet at δ 9.50 ppm from TMS may be due to the hydrogen at position 11 of cryptolepine (H-11) which is the only H without neighbouring hydrogen atoms to split its signal.

Table: 3 Interpretation of NMR spectrum of SA-I

S.no Delta value ppm Position of Hydrogen atom Type of peak
1 5.83 H-5 Singlet
2 8.27 H-6 Aromatic multiplet
3 9.50 H-11 Singlet aromatic proton & isolated aromatic proton.

Figure: 4 13 C NMR Spectrum of isolated SA-I

Interpretation of <sup>13</sup>C NMR spectrum of isolated SA-I

Hetero aromatic carbons give signals within the range δ 100-165ppm andnine peaks within this range of chemical shift under CH 13C DEPT analysis of isolated SA-I. This confirms the nine protonated hetero aromatic carbons in SA-I: C-1,2,3,4,6,7,8,9, and 11.sp3 hybridised carbons absorb at lowest field (δ 0-50ppm) as compared to sp2(δ100-160ppm aromatic) and sp (δ 65-90ppm) hybridised carbons. Isolated SA-1 showed a singlet at δ 40.3ppm which confirms the presence of the only methyl carbon on the quinoline nitrogen at position 5 of SA-I ( >N-CH3).

Table: 4 Interpretation of 13 C NMR spectrum of Isolated SA-I

S. No Delta value ppm Types of Carbon
1 100-165 Hetero aromatic carbon
2 0-50 Hybridised carbon
3 100-160 Aromatic carbon
4 40.3 Methyl carbon

Figure: 5 Mass spectrum of isolated SA-I

Interpretation of Mass spectrum of isolated SA-I

The base peak of mass spectrometric analysis of isolated SA-Iwas found to be at m/z Value of 248.07 corresponding to 100% relative intensity. Hence, the base peak of the mass spectrum of the isolated SA-I which also corresponds to the molecular ion actually occured at m/z 248.07. This value is in agreement with the literature value of the molecular weight of SA-I which was 232g. The peak occuring at m/z 220.20(29.36%).might correspond to N-demethylated fragment of SA-I.

Table: 5 Interpretation of Mass spectrum of SA-I

S. No Type of ion m/z Value
1. Molecular ion 248.07
2 Methylene fragment 234.13
3 Demethylated fragment ion 220.20
STRUCTURE OF SA-I

IUPAC NAME : 5-methyl 5-H indolo [3,2-b]quinoline.(hydrated cryptolepine)

Molecular Formula: C16H12N2

Molecular Weight : 248.286g/mol

 DETERMINATION OF ANTIMYCOBACTERIAL ACTIVITY OF HYDRO ALCOHOLIC EXTRACT OF <italic>Sidaacuta</italic>Burm (Leaves) (HAESA) by Luciferase reporter phage assay

The antimycobacterial effect determined for the hydro-alcoholic extract of SidaacutaBurmand isolated SA-I as per (Papitha.net al 2013)[38]

Figure :6 Antimycobacterial effect of Hydro-alcoholic extract of Sidaacuta burm (leaves) (HAESA)

Table: 6 Determination of Antimycobacterial activity of Hydro-alcoholic extract of Sidaacuta Burm leaves against standard strain of Mycobacterium tuberculosis H37Rv.

S.No Compound Concentration(µg/ml) Relative light unit value % of Inhibition
1 Isolated alkaloid SA-I 500 12715 0%
1000 12596 0%
2 HAESA 500 110 99%
1000 75 99%
3 Isoniazid 1 792 92%
4 Rifampicin 1 299 96%

The percentage inhibition of Sidaacuta Leaf (HAESA) against mycobacterium tuberculosis standard strain of H37Rv. HAESA (500µg/ml and 1000µg/ml) showed anti mycobacterial effect was found to be 99% in comparison with isoniazid (1ug/ml) 92% and rifampicin (1µg/ml) 96%.

The isolated compound (SA-I) showed the spectral data of cryptolepine. The percentage inhibition of hydro alcoholic extract of Sidaacuta (500 & 1000µg/ml) against mycobacterium tuberculosis showed antimycobacterial effect was found to be 99% in comparison with isoniazid (1ug/ml) 92% and rifampicin (1µg/ml) 96%, whereas isolated cryptolepine did not any sensitiveness towards mycobacterium tuberculosis.This research reports cryptolepine did not show any antimycobacterial effect. It indicated that antimycobacterial effect of hydro-alcoholic extract of Sidaacuta may be due to the combined nature of cryptolepine with other phytoconstituents.

 Conclusion

The present research draws the conclusion that hydroalcoholic extract of Sidaacutaplant showed extreme antimycobacterial effect, which may be due to the combined nature phytoconstituents.

Acknowledgement

We are grateful to our respected Dean. Dr. D. MARUDHUPANDIAN, M.S., FICS., FAIS, Madurai Medical College, Madurai, for providing these research facilities to carry out this work

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