Phytochemical Evaluation of Tribal Drug Veerasingam Pattai

Zanthoxylum tetraspermum Wight and Arn stem bark

 

V.R. Ravikkumar¹*, V. Gopal², N. Ravichandaran³, P. Brindha³ and T. Sudha⁴

¹Research Scholar, CARISM, Sastra University. Thanjavur – 638402, Tamil Nadu, India.

²Mother Theresa Post Graduates and Research Institute of Health Sciences, Pudhucherry - 605006, India.

³CARISM, Sastra University. Thanjavur – 638402, Tamil Nadu. India.

^1,4The Erode College of Pharmacy, Erode- 638112, Tamil Nadu, India.

*Corresponding Author E-mail: ravisrkumar@yahoo.com

 

Zanthoxylum tetraspermum Wight and Arn stem bark (Rutaceae) known as Veerasingam pattai is traditionally used by the tribals for treating tooth ache and in tooth extraction. The present study reveals the Phytochemical Evaluation of Tribal Drug Veerasingam Pattai by HPTLC and LC-MS/MS, which is used for treating microbial infections and tooth ache.  The preliminary screening of Phytochemicals and HPTLC profile of secondary metabolites from the stem bark of zanthoxylum tetraspermum Wight and Arn have shown the presence of various important secondary metabolites such as alkaloids, flavonoids, glycosides, Phenols, saponin,  sterols, tannins and triterpeniods. Ethanolic and aqueous extracts were selected for LC-MS/MS analysis due to high percentage yield. In the LC-MS/MS analysis, bioactive Phytochemical Compounds like Dihydronitidine, Acetonyl Dihydro avicine, Sesamin, 8-O-Desmethyl-N-nornitidine, Oxyavicine, Dihydroacvine, Liriodenine and Savinin were identified in the Ethanolic extract and N-caffeoyl O-methylyramine, Bergenin, Tetrahydrocurcumin, Tetrahydropalmatine, Sesamolin, Fagaridine and  8-O-Desmethy-N- nornitidine were identified in Aqueous extract of Veerasingam Pattai. The generated data has been provided the basis for its therapeutic value and can be used as therapeutant.

 

INTRODUCTION:

Zanthoxylum tetraspermum Wight and Arn., (Rutaceae) is a thorny, stout, aromatic, climbing shrub, with brown bark having short recurved prickles, found in Western ghats especially in Nilgiri, Aanaimalai and in Eastern ghats Kolli hills at the altitudes of 1,200-1,800 and also found in western ghats of Kerala and Karnataka. The wood is yellowish and soft. The plant is credited in Srilanka with stimulant, astringent and digestive properties and is prescribed in dyspepsia and diarrhoes^1-4. Two benzophenathrene like 8-actonyldihydronitidine, 8-actonyldihydroavicine, liriodenine, sesamin, lichexanthone, (+)-piperitol gamma-gamma-dimethylallylther, savinine, α - amyrin, iso-arborenol, betulinic acid, β-sitosterol, Spathulenol have been reported⁵. This plant is ethno medically reputed and is used in the treatment of various ailments. A preliminary ethno medical survey revealed that this plant was used by the tribals in tooth extraction.  Literature survey revealed that the plant was scientifically under explored. 

 

Hence this plant was selected for the present study.  The present research was carried out to evaluate the Phytochemical potential of the tribal drug “Veerasingam pattai” ^1-5.

 

MATERIALS AND METHODS:  

The stem bark of Z.tetraspermum were collected from Cholakkadu, a village of  Kolli hills, 60 km away from Namakkal (Tamilnadu) in the  month of August (2007). The plant was identified by local people of that village and authenticated by G.V.S. Murthy, Join Director, Botanical Survey of India (BSI), Southern Circle, Coimbatore, (No. BSI/SC/5/23/ 07-08/ Tech 715). A herbarium specimen of the plant was preserved in the Department of Pharmacognosy, The Erode College of Pharmacy, Erode, used for further references.

 

Chemicals:

All the reagents used were of analytical grade obtained from S.D. Fine chemicals Ltd, Mumbai. HPLC grade solvents were obtained from Merck India limited, Mumbai, India.

 

Extractions:

The stem bark of Zanthoxylum tetraspermum were washed with water, Air- dried at room temperature and reduced to coarse powder. The coarsely powdered (250 gm) dried stem bark of Z. tetraspermum was subjected to soxhelt extraction with different solvents ranging from non-polar to polar solvents like petroleum ether (40-60^o), chloroform, ethyl acetate, ethanol and water ⁵. The extracts were concentrated by in vacuum, weighed the extracts and calculated the extract values in percentage with reference to the air dried drug (1.12,1.37,1.96,3.75,4.06% w/w respectively). The freshly prepared extracts were chemically tested for the presence of different Phytoconstituents using standard      methods^5-7.

 

Phytochemical analysis by HPTLC:

The different extracts of Veerasingam Pattai (Z. tetraspermum) were dissolved in appropriate solvents and centrifuged at 3000 rpm for 5min.  These solutions (mg/ml) were used as test solution for HPTLC analysis^8-10. 2µl of test solutions were loaded as 5mm band length in the 3 x 10 Silica gel 60F₂₅₄ TLC plates using Hamilton syringe and CAMAG LINOMAT 5 instrument. Different extracts of Veerasingam Pattai  (Z. tetraspermum) (Petroleum ether, Chloroform, Ethyl acetate, Ethanol   and Aqueous) were loaded and plates were kept in TLC twin trough developing chamber (after saturation) with respective mobile phases are Petroleum ether-Chloroform (2:8), Petroleum ether-Chloroform (2:8), Chloroform-Methanol (8:2), Chloroform-Methanol (8: 2)and Petroleum ether-Chloroform-Methanol (3: 3: 4) and the plates were developed up to 90 mm. The developed plates were dried by hot air.The plates were kept in Photo-documentation chamber (Camag Reprostar 3) and captured the images at visible light, UV 254 nm and UV 366 nm. The developed plates were sprayed with Anisaldehyde sulphuric acid spraying reagent and dried at 105°C in Hot air oven. After derivatization of the plates were fixed in scanner stage (Camag TLC Scanner 3) and scanned at visible light and UV 366 nm. Peak table, Peak display and Peak densitogram were noted by using win CATS 1.3.4 version software. Fingerprinting profile showed numerous bands, which indicated the number of compounds present in the samples.

 

Table 1. Preliminary Phytochemical analyses of Z. tetraspermum stem bark

S.No.	Phytoconstituents	Pet. Ether Extract	Chloroform Extract	Ethyl  acetate Extract	Ethanolic Extract	Aqueous  Extract
1	Alkaloids	+	+	+	+	+
2	Carbohydrates	-	-	-	-	-
3.	Glycosides	-	-	-	+	+
4	Sterols	+	+	-	-	-
5	Fixed oils and fats	+	+	+	-	-
6	Triterpenoids	+	+	+	+	+
7.	Tannins-phenolic compounds	-	-	-	+	+
8.	Proteins and amino Acids	-	-	-	-	+
9.	Saponins	-	-	-	+	+
10	Gums and  Mucilages	-	-	-	+	+
11	Flavones and Flavonoids	-	-	-	-	+

(+) Denotes presence, ( –) Denotes absence

 

Figure 1. Track A and B – HPTLC pattern of Petroleum ether and Chloroform extracts of  stem bark of   “Zanthoxylum tetraspermum Wight and arn” Peak densitogram display (Scanned at 254nm)

 

Figure 2. Track C and D – HPTLC pattern of Ethyl acetate and Ethanolic extracts stem bark of  “ Zanthoxylum  tetraspermum Wight andarn” Peak densitogram display (Scanned at 254nm)

Phytochemical analysis by LC-MS/MS

The principle of LC-MS/MS is based on the fragmentation of charged ions and the detection of the resulting fragments. Thus it has a much higher selectivity and sensitivity than LC-MS and makes it possible to elucidate e.g. metabolite structures.

 

The ethanolic and aqueous extracts of stem bark of Z. tetraspermum were dissolved in appropriate solvents (mg/ml) used for LC-MS/MS analysis. The compounds were identified with the help of M/Z ratio value^7-14.

 

Chromatographic condition

Analyses were performed by using; UHPLC + Focused, Dionex 300 Series; (Germany), UV detection at 361 .0nm. The stationary phase was used RP-C₁₈ Acclain 120. Mobile Phase A: Acetonitrile and Mobile Phase B: 0.1% Formic acid in Water was used mobile phase in gradient technique. Injection volume was 50 ml.(0 min = 7% A, 93% B; till 5 min 7%A, 93% B; then reaches to 70%, 30% B; in another 15 min; reaching to 7% A, 93% B in 5 min)

 

MS/MS Condition

Spectroscopic analyses were performed by using MICROTOF Q II (Bruker Germany) ESI (Electron spray ionization) Ionization Mode was used. Quadruple mass Analyzer and Time of flight detector were used. Inlet nitrogen gas pressure of 30 psi, 6.0/min; Positive polarity; -4500 V, scan from 50-1500 m/z; MSMS mode Auto Msn, Collision Energy at 15 eV; Data analysis through Mass Bank, Metlin Scripps soft wares and other literatures were used ^{11- 33}.

 

 

Figure 3. Track E– HPTLC pattern of aqueous extract of stem bark of “Zanthoxylum tetraspermum Wight and Arn” Peak  densitogram display (Scanned at 254nm)

 

 

Table 2. Phyto constituents identified by LC-MS/MS in ethanolic extract of Z. tetraspermum stem bark

S.No	Name of the compounds	Molecular Formula	MW	M/Z Value	Compound Nature	**Activity
1.	Dihydronitidine	C21H19NO4	349	350.1	Alkaloid	Anticancer .,Antimicrobial
2.	Acetonyl dihydro avicine	C23H19NO5	389	390.2	Alkaloid	Antioxidant, Antileukemic , Antimicrobial
3.	Sesamin	C20H18O6	354	355.1	Lignan	Anticholestermic,  Antihypertensive, Antioxidant
4.	8-O-Desmethyl- N-nornitidine	C19H13NO4	319	320.1	Alkaloid	No activity reported
5.	Oxyavicine	C20H13NO5	347	348.1	Alkaloid	Analgesic , AntiInflammatory
6.	Dihydro avicine	C20H15O4	333	334.1	Alkaloid	No activity reported
7.	Liriodenine	C17H9NO3	275	276.1	Alkaloid	Muscarnic Activity, Calcium channel blocker, Antimicrobial
8.	Savinin	C20H16O6	352	353.1	Lignan	Antitumor, Spermicidal

 

Table. 3. Phytoconstituents identified by LC-MS/MS in aqueous extract of  Z. tetraspermum stem bark

S. No	Name of the compounds	Molecular Formula	MW	M/Z Value	Compound Nature	**Activities
1.	N-caffeoyl O-methylyramine	C18H19NO4	313	314.2	Amide	Tonics
2.	Bergenin	C14H16O9	328	328.7	Isocoumarin	Hepatoprotective, Antiarrhythmic, Antiulcer
3.	Tetrahydrocurcumin	C21H24O6	372	372.7	Curcuminoid	AntiInflammatory, Antioxidant, Hepatoprotective
4.	Tetrahydropalmatine	C21H25NO4	355	356.2	Alkaloid	Analgesic, Muscle Relaxant, Sedative
5.	Sesamolin	C20H18O7	370	371.3	Lignan	AntiInflammatory, Antioxidant
6.	Fagaridine	C20H17NO5	351	352.1	Alkaloid	Antitumor, AntiMalarial
7.	8-O-Desmethyl-N-nornitidine	C19H13NO4	319	320.7	Alkaloid	No activity Reported

Figure 4.  LC-MS/MS Spectrum of ethanolic extract of Z. tetraspermum stem bark

 

 

Figure 5. MS-MS spectrum of different identified compounds of ethanolic extract of stem bark of Z. tetraspermum wight  and Arn

Figure 6.  LC-MS/MS Spectrum of Water extract of Z. tetraspermum stem bark

 

Figure.7 MS-MS spectrum of different identified compounds of aqueous extract of stem bark of Z. tetraspermum wigh and Arn

RESULTS AND DISCUSSION:

The medicinal plants are having various secondary metabolites such as alkaloids, Flavonoids, glycosides, phenols, tannins, saponins, sterols etc. The preliminary phytochemical screening tests and HPTLC profile may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development [34].  Further, these tests facilitated their quantitative estimation and qualitative separation of the pharmacologically important active phytochemical components. The extract of Veerasingam Pattai  (Z. tetraspermum) have revealed the presence of alkaloids, flavonoids, glycosides, lignans, phenols, tannins, triterpeniods sterols, saponins, fats (Table 1) and oils etc.  HPTLC fingerprint profile showed numerous bands which indicated the number of compounds present in the different extracts of Veerasingam Pattai (Z. tetraspermum) (Figure.1-3). Among the various extracts, ethanolic and aqueous extracts were known to be rich in the phytochemicals as well as high percentage yield (3.76 and 4.06% w/w respectively). HPTLC fingerprint profile of ethanolic and aqueous extract of Veerasingam Pattai    (Z. tetraspermum) showed more number of bands and high Peak area densitogram display (Figure. 1-3) and also it has been more traditional value. So that, ethanolic and aqueous extracts of Veerasingam Pattai (Z. tetraspermum) were selected for identification of bioactive compounds by the analysis of LC-MS/MS technique. The following pharmacologically active compounds were identified with the help M/Z ratio value.  The compounds like Dihydronitidine 1, Acetonyl dihydro avicine 2, Sesamin 3, 8-O-Desmethyl- N-nornitidine 4, Oxyavicine 5, Dihydro avicine 6, Liriodenine 7 and Savinin 8 were identified from ethanolic extract and N-caffeoyl O-methylyramine 1, Bergenin 2, Tetrahydrocurcumin 3, Tetrahydropalmatine 4, Sesamolin 5, Fagaridine 6 and  8-O-Desmethyl-N-nornitidine 7 were identified from aqueous extract of  tribal drug Veerasingam Pattai  (Z. tetraspermum stem bark) ^11-33 (Table 2- 3, Figure 4-7).

 

Two benzophenanthridine alkaloids like Fagaridine, 8-O-Desmethyl -N-nornitidine, oxyavicine, Dihydroavicine and N-caffeoyl –O -methyltyramine (amide), Bergenine (coumarin), Tetra hydo curcumin (curcuminoid), Tetra hydropalmatine (Alkaloid), sesamolin (lignan) are described for the first time in Z. tetraspermum Wight and Arn^11-33.

 

Alkaloids are one of the important classes of secondary metabolites which are heterocyclic indole compounds which have shown to contain pharmacological properties such as hypotensive, anticonvulsant, antiprotozoal, antimicrobial and antimalarial, analgesics, stimulants, anesthetic, hallucinogens and antibacterial activities³⁴.

 

Flavonoids are the phenolic substances which are the largest group of phenols. They generally occur as a C₆-C₃ unit linked to an aromatic ring. They are other plant constituents with antibacterial and antifungal properties. They are used in treating stomach ulcer and inhibit HIV-1 integrase and HIV-1 protease enzymes which are responsible for the HIV replication ³⁴.

 

Commonly the antimicrobial activities of the medicinal plants may be related to the presence of glycosides and saponins. The presences of glycosides are reported to possess strong anti-bacterial activities and exist as antibiotics ³⁴.

 

Plants have limitless ability to synthesize phenols or their derivatives. The presence of phenols in all types of tissues is a characteristic feature of plants. Steroidal compounds are of importance and interest in pharmacy due to their relationship with such compounds as sex hormones. They are given to breast feeding mothers to ensure their hormonal balance, since steroidal structure could serve as potent starting material in synthesis of these hormones ³⁵.

 

Saponins are glycosides of both triterpenes and sterols generally possessing five sugar units and gluconic unit as a component. The occurrence of saponins has been reported in over seventy families of higher plants. They are used to reduce body cholesterol by preventing its re-absorption³⁵.

 

The identified secondary metabolites from Veerasingam Pattai (Z. tetraspermum Wignt and Arn) provided the chemical basis for its pharmacological action and traditional use.

 

CONCLUSION:

The study has revealed the presence of secondary metabolites of various therapeutically active compounds in the Veerasingam Pattai (Stem bark extract of Z. tetraspermum Wight and Arn).by the analysis of HPTLC and LC-MS/MS. It has further confirmed that the bark extract could be used for the treatment of various dental infections. The results credence to the folkloric use, if this plant in treating (dental infection) and showed that Veerasingam Pattai (Zanthoxylum tetraspermum Wight and Arn) could be exploited for new potent dental care drugs.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr.  P. Brindha, Head, CARISM, K.P Arun.  Ph.D. Research Scholar, CARISM, SASTRA University, Thanjavur for motivation and support to carry out LC-MS/MS study.

 

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Accepted on 28.09.2013      © RJPT All right reserved