Phytochemical Evaluation and Chromatographic Fingerprint Analysis on Flavonoids Compounds in Leucas aspera (Willd.) Link Leaf by HPTLC

Muthupandi Annapandian, Shanmuga Sundaram, and Gomathi: Phytochemical Evaluation and Chromatographic Fingerprint Analysis on Flavonoids Compounds in Leucas aspera (Willd.) Link Leaf by HPTLC

Authors

INTRODUCTION

Leucas aspera (Willd) Link (L. aspera) belongs to Lamiaceae (Labiatae) family. It is a common weed in India and widely distributed throughout the country. The leaves of L. aspera are obtuse, linear or linearly lanceolate or petiolate. The length of the leaf is about 8.0 cm and 1.25 cm in broad. Though L. aspera has many vernacular names depending on the region, most commonly known as Thumbai.1 The juice of L. aspera leaves are used as remedy for chronic skin eruptions, chronic rheumatism and psoriasis.2 In villages L. aspera leaves are used as insecticides and mosquito repellent. Flavonoids are the phenolic compounds that comprise in large group of plants and widely distributed throughout the plant kingdom. Several studies have suggested that flavonoids are effective in prevention and treatment of many diseases.3,4 In our present study, we attempted to evaluate the presence of flavonoids content and fingerprinting profile of flavonoids present in the L. aspera leaves by HPTLC technique using different solvent extracts.

MATERIALS AND METHODS

Plant materials

The plant L. aspera was collected from southern part of Tamil Nadu (Kandappakottai, Dindigul District) and was authenticated by Prof. P Jayaraman, Director, Institute of Herbal Botany and Anatomy Research Centre, Tambaram, Chennai (PARC/2014/2272). The leaves were then isolated, cleaned and dried under sun shadow. Dried leaves were crushed to powder and passed through pharmaceutical sieve no 40 and 80. Any materials which retained at sieve-80 were collected and used for extraction.

Preparation of crude extracts

Five different solvents were used for extraction process with increase in polarity, which included petroleum ether (60-80°C), ethyl acetate, chloroform, ethanol (70%) and isopropyl alcohol. Soxhlet apparatus was used for petroleum ether extraction. Six hundred g of powdered leaf was extracted with 2.5 L of petroleum ether (60-80°C). Excess solvent was removed by distillation method. Crude extract was dried at vacuum desiccators and stored in a glass bottle at -20°C for further studies. All other solvent extracts were prepared by cold maceration technique. Weighed powder were soaked in individual solvents viz., ethyl acetate (200 g + 1.0 L), chloroform (200 g + 1.0 L), isopropyl alcohol (200 g + 1.0 L) and ethanol 70% (600 g + 2.5 L) in a separate round bottom flask for 72h with occasional shaking. After soaking, the mixture was heated at 30±5°C for 60 min. Heated solution was cooled to room temperature and filtered through muslin cloth and Whatman filter paper. Excess solvents were then removed by distillation method and allowed to evaporate at room temperature. Dried crude extracts were collected and stored in an individual glass bottles at -20°C for further studies.

Qualitative phytochemical screening of flavonoids

All the five solvent extracts were screened for the presence of flavonoids with all respective testing procedures include shinoda’s test, alkaline reagent test, lead acetate test and sulphuric acid test as described in the textbook by Harborne JB (eds).5

Quantitative estimation of total flavonoid compounds

Total flavonoids were estimated by using spectrophotometric technique.6 Flavonoids give yellow color when treated with aluminum chloride reagent (1% w/v) and this principle was used in flavonoid estimation. Rutin was used as standard in methanol solution (100 mg/ml). Test sample was prepared with methanol to the final concentration of 100 mg/ml. One ml of test and standard solutions were taken in a separate 10 ml volumetric flask and 1.0 ml of aluminum chloride reagent was added to both test and standard solutions. Volumes were made up to 10 ml with methanol and optical density measured at 410 nm against blank solution exactly at 15 min after adding aluminum chloride reagent.

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-942_img_0.jpg

T -- sample (test) solution; S -- standard solution; TB -- sample blank; SB -- standard blank; T – TB -- absorbance of test; S – SB -- absorbance of standard *Subtract the corresponding blank values from the standard and test absorbance values (S – SB; T – TB). Calculate the flavonoid content with respect to Rutin and express as % w/w of flavonoids.

Thin layer chromatography (TLC) Studies

Thin Layer Chromatography has been used for isolation of compounds form the crude extracts. Pre-coated silica gel on aluminum plates (Merck pre-coated Al. Sheets - 1.05554) were used as adsorbent for elution. Crude extracts were dissolved in HPLC-grade methanol to the final concentration of 10 mg/mL. Based on trial and error method toluene: ethyl acetate: acetic acid (55: 45: 1) was used as mobile phase (eluting agent). Spots were observed at 254 nm and 366 nm.

HPTLC finger printing profile of flavonoids

For fingerprinting studies, a densitometric high performance thin layer chromatographic (HPTLC) system was used. The HPTLC system equipped with Shimadzu flying spot densitometer CS9301, Camag TLC Scanner-3 with win Cats Planar chromatography manager, Applicator CAMAG - Linomat IV and Linomat V and saturated developing chamber CAMAG Twin trough 20x20 cm was used. HPLC grade Methanol was used as a solvent for all the extracts solution (10 mg/mL). Spots were made on a pre-coated plate {Silica gel 60 F254: (Merck pre-coated Al. Sheets - 1.05554)} with an aid of sampling machine, and solvent front run up to 9 cm height. Ethyl acetate: acetic acid: formic acid: water (100:11:11:10) was used as a mobile phase and 1% aluminum chloride solution was used as spraying agent for flavonoids profiling. Rutin was used as a standard reference.

RESULTS

Phytochemical studies

The present study was carried out on the crude extracts of petroleum ether, ethyl acetate, chloroform, ethanol and isopropyl alcohol obtained from leaves of L. aspera. Petroleum ether, isopropyl alcohol and ethanol extracts had shown to strong positive for flavonoids test than chloroform and ethyl acetate extracts Table 1. Quantitative estimation showed the higher concentration of flavonoids present in the ethanol extract compared to other solvent extracts. The summary of amount of total flavonoids content present in each extract was described in Table 2.

Table 1

Preliminary pytochemical screening of flavonoids present in crude extracts of L. aspera leaves.

Test for flavonoidsPetroleum ether extractEthyl Acetate extractChloroform extractIsopropyl alcohol extractEthanol extract
Shinoda’s Test++++++++
Alkaline reagent test+++++++
Lead acetate test+++++++
Sulphuric acid test++++++

[i] + Positive. ++ Strong positive.

Table 2

Summary of total flavonoids compounds present in L. aspera leaf extracts.

Leucas aspera leaf extractsTotal flavonoids content (g)
Petroleum ether extract13.22
Ethyl Acetate extract14.90
Chloroform extract13.22
Isopropyl alcohol extract13.39
Ethanol extract15.97

[i] * all values are expressed in g per 100g of extract.

Chromatographic studies

HPTLC studies showed blue, greenish blue and yellowish green coloured fluorescent zones at UV 366 nm after derivatization. These fluorescent zones confirmed the presence of flavonoids in the L. aspera leaf extracts Figure 1. The order of presence of flavonoids (fluorescent zone) in L. aspera extract was ethanol > isopropyl alcohol > ethyl acetate > chloroform > petroleum ether. The Rf value of the extracts were found to be 0.61, 0.66, 0.67, 0.70, 0.76 and 0.77 of the peaks. Out of 10 peaks 6 were identified as flavonoid compounds. The complete details of Rf values and area of the peaks were presented in Table 3. Rutin was used as the standard compound for flavonoid identification.

Figure 1

HPTLC profile of L. aspera leaves under UV 254 and 366 nm.

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-942_img_2.jpg
Table 3

Peak table with RF values, height and area of flavonoids and unknown compounds present in L. aspera leaf extracts.

TrackPeakRfHeightAreaAssigned substance
Rutin10.67609.521679.7Rutin
Rutin20.7731.1598.4Unknown
PELA10.6617.1329.1Flavonoid
PELA2---Unknown
EALA1---Unknown
CFLA1---Unknown
IALA1---Unknown
IALA20.7033.0983.9Flavonoid 1
IALA30.7636.7871.6Flavonoid 2
EELA10.6146.21575.8Flavonoid 1
EELA20.6663.71802.6Flavonoid 2
EELA30.67165.75368Flavonoid 3

DISCUSSION

Preliminary phytochemical studies give a broad idea to researchers for their further research. In this present study, preliminary phytochemical screening confirmed the presence of flavonoids in the L. aspera leaf extracts. Out of which, petroleum ether (non-polar), isopropyl alcohol (polar) and ethanol (polar) extracts had shown strong positivity. The quantitative estimation revealed, total flavonoid compounds are almost equally present in the L. aspera leaf extracts. But the HPTLC fingerprint profiles showed, among the five solvent extracts, flavonoids were predominantly found in ethanol extract of L. aspera followed by isopropyl alcohol, ethyl acetate, chloroform and petroleum ether extracts. At least two types of flavonoids were observed in each extract Figure 2 and 3. Four unknown compounds were observed by HPTLC fingerprint profile of flavonoids in different solvent extracts of L. aspera leaves. The summary of HPTLC chromatogram of different solvent extracts of L. aspera is presented in Table 4. Our study findings are comparable with the existing literature where they used methanol extract of whole plant of L. aspera.7 Flavonoids have potent antioxidant properties and it can modulate the various enzymatic activities due to their interaction with various biomolecules.8 Traditionally L. aspera was used as insecticide and antipyretic. Several studies showed L. aspera has a potential anti-diabetic effect9-11, larvicidal effect 12-14 and antimicrobial effect.15-17

Figure 2

HPTLC chromatogram of L. aspera leaf extracts: All tracks at wavelengths.

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-942_img_3.jpg
Figure 3

HPTLC chromatogram of different solvent extracts of L. aspera leaf.

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-942_img_4.jpg
Table 4

HPTLC fingerprint profile of flavonoids present in different solvent extracts of L. aspera leaf.

Rf valueRutinPetroleum etherEthyl acetateChloroformIsopropyl alcoholEthanol
0.61-----+
0.65--+---
0.66---+-+
0.67++----
0.70----+-
0.74------
0.76----++
0.77++----

CONCLUSION

In recent years the usage of Indian Medicine system and Traditional Medicine systems are increased. Many people have started using alternative medicines particularly in chronic disorders. Herbals and phytocompounds based medicines are believed to be less toxic and effective. Leucas aspera is a good source for flavonoid compounds and seems to have promising phytocompounds that can be used to alleviate many chronic illnesses with its isolated active compounds. Further studies are required for identification and characterization of flavonoid compounds present in the Leucas aspera leaves, which is potential for therapeutic applications.

Acknowledgements

None

Notes

[3] FINANCIAL SUPPORT AND SPONSORSHIP Nil

[4] Conflicts of interest CONFLICTS OF INTEREST All authors have no conflict of interest.

ABBREVIATIONS USED

CFLA

Chloroform extract of Leucas aspera

EALA

Ethyl acetate extract of Leucas aspera

EELA

Ethanol extract of Leucas aspera

g

gram

HPLC

High performance liquid chromatography

HPTLC

High performance thin layer chromatography

IALA

Isopropyl alcohol extract of Leucas aspera

L

Litre

L. aspera

Leucas aspera (Willd) Link

PELA

Petroleum ether extract of Leucas aspera

TLC

Thin layer chromatography

REFERENCES

1 

Rai V, Agarwal M, Agnihotri A, Khatoon S, Rawat A, Mehrotra S. Pharmacognostical Evaluation of Leucas aspera Link. Nat Prod Sci. 2005;11(2):109–14

2 

Kirtikar KR. Indian Medicinal Plants. Vol. III(2)Sec ed. Mohan Basu Lalit, editor. Allahabad, India: p. 1593

3 

Middleton E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacological Rev. 2000;52(4):673–751

4 

Ren W, Qiao Z, Wang H, Zhu L, Zhang L. Flavonoids: promising anticancer agents. Med Res Rev. 2003;23(4):519–34

5 

Harborne J , editor. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. Third ed. Springer Science and Business Media; London: 1998. p. 334

6 

Chang C-C, Yang M-H, Wen H-M, Chern J-C. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002;10(3):178–82

7 

Karthikeyan S, Sivakumar A, Anbalagan M, Nalini E, Gothandam KM. Finger Printing of Alkaloids, Steroids and Flavonoids using HPTLC of Leucas aspera L.Whole Plant ethanolic Extract. J. Pharm. Sci. and Res. 2013;5:67–71

8 

Catopano AL. Antioxidant effect of flavonoids. Angiology. 1997;48(1):39–46. (44).

9 

Mannan A, Das H, Rahman M, Jesmin J, Siddika A, Rahman M, et al. Antihyperglycemic activity evaluation of Leucas aspera (Willd.) Link leaf and stem and Lannea coromandelica (Houtt.) Merr. bark extract in mice. Adv Nat Applied Sci. 2010;4(3):385–8

10 

Gupta N, Agarwal M, Bhatia V, Sharma RK, Narang E. A comparative anti-diabetic and hypoglycaemic activity of the crude alcoholic extracts of the plant Leucas aspera and seeds of Pithecellobium bigeminum in rats. IJRAP. 2011;2:275–80

11 

Tukaram T, Parvati CV, Venkata-Rao N, Patil P. Srinivas-Rao N. Evaluation of the extracts of Leucas aspera on biochemical profiles in experimental model of diabetes mellitus (Type- I) in rats. Inter Res J Pharm. 2011;2(3):246–8

12 

Suganya G, Karthi S, Shivakumar MS. Larvicidal potential of silver nanoparticles synthesized from Leucas aspera leaf extracts against dengue vector Aedes aegypti. Parasitol Res. 2014;113(3):875–80

13 

Elumalai D, Hemavathi M, Hemalatha P, Deepaa CV, Kaleena PK. Larvicidal activity of catechin isolated from Leucas aspera against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res. 2016;115(3):1203–12

14 

Kovendan K, Murugan K, Vincent S, Barnard DR. Studies on larvicidal and pupicidal activity of Leucas aspera Willd. (Lamiaceae) and bacterial insecticide, Bacillus sphaericus, against malarial vector, Anopheles stephensi Liston. (Diptera: Culicidae). Parasitol Res. 2012;110(1):195–203

15 

Rahman MA, Islam MS. Antioxidant, antibacterial and cytotoxic effects of the phytochemicals of whole Leucas aspera extract. Asian Pac J Trop Biomed. 2013;3(4):273–9

16 

Chew AL, Jessica JJA, Sasidharan S. Antioxidant and antibacterial activity of different parts of Leucas aspera. Asian Pac J Trop Biomed. 2012;2(3):176–80

17 

Mangathayaru K, Lakshmikant J, Shyam Sundar N, Swapna R, Grace XF, Vasantha J. Antimicrobial activity of Leucas aspera flowers. Fitoterapia. 2005;76(7):752–4

ABOUT AUTHORS

Vellaichamy M. Annapandian, is a Consultant, Academic Research, Narayana Hrudayalaya Foundations, Bangalore, India. He has published more than 10 peer review research articles, 1 book chapter and 12 conference proceedings. The author’s work has been published in medical, pharmacology and phytopharmacy journals.

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-942_img_6.jpg