Elemental Analysis in Piper betle Linn. and Jatropha gossypifolia Linn. leaves: Biosafety Studies

 

Sapna Saini^1*, Sanju Nanda², Anju Dhiman³

¹Research Scholar, Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, Haryana, India.

²Professor, Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, Haryana, India.

³Assistant Professor, Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, Haryana, India.

 

ABSTRACT:

Heavy metal toxicity is a primary matter of concern due to ecological and nutritional reasons. Determination of minerals and heavy metal concentration in herbal remedies is crucial to assure their biosafety. The present research study was designed to estimate the concentration of some trace elements v.i.z. Copper (Cu), Zinc (Zn), Magnesium (Mg), Calcium (Ca) and heavy metals v.i.z. Cadmium (Cd), Arsenic (As) and Mercury (Hg) in Piper betle L. and Jatropha gossypifolia L. dried leaf powder. The dried leaf powder of selected medicinal plants was wet digested using nitric acid, hydrogen peroxide and hydrochloric acid as specified. The quantitative estimation of trace elements and heavy metals was done using atomic absorption spectrophotometer (AAS) by plotting standard calibration curve of different metals. Results are expressed as mean ± standard deviation and analyzed by student’s‘t’ test. Values are considered significant at P < 0.05. The order of concentration of metals in P. betle L. dried leaf powder was found to be: Mg > Ca > Zn > Cu > As > Cd = Hg and in J. gossypifolia L. dried leaf powder was found to be: Ca > Mg > Zn > Cu > Cd = As = Hg. The concentration of trace elements (Cu, Zn, Ca, Mg) was found to be within limit and for heavy metals (Cd, As, Hg) below the detection limit when compared with standard values. On this basis, it can be concluded that both plant materials were found to be safe.

 

 

 

INTRODUCTION:

Heavy metals are predominant environmental pollutants and their increasing concentration causes toxicities which impose significant negative impact on our ecological, evolutionary, nutritional and environmental system. Heavy metals toxicity in the environment occurs either by natural means or through human activities. Various sources of heavy metals include soil erosion, natural weathering of the earth’s crust, mining, industrial effluents, smelters, coal burning plants, urban runoff, sewage discharge, pesticides and fertilizers applied to crops etc.¹

 

 

There are approximately fifty elements from transition metals, metalloids, lanthanides and actinides series which are considered as heavy metals. Among them lead (Pb), mercury (Hg), arsenic (As), and cadmium (Cd) are generally considered as non-essential heavy metals. They all four are leader toxic metals that can cause poisoning even at trace level. However, metals such as iron (Fe), chromium (Cr), copper (Cu), zinc (Zn), cobalt (Co), nickel (Ni), selenium (Se) and bismuth (Bi) etc. are considered as essential metals and are known to play a vital role in physiological concentrations but they can also be toxic in larger doses^{2, 3}. Excessive intake of heavy metals can cause serious health hazards such as chronic exposure to As can lead to dermal lesions, skin cancer, peripheral neuropathy, and peripheral vascular disease, while chronic ingestion of Cd can have adverse effects such as prostatic proliferative lesions, bone fractures, kidney dysfunction, hypertension lung cancer, and pulmonary adenocarcinomas. Excessive intake of Pb can damage the skeletal, circulatory, nervous, enzymatic, endocrine, and immune systems⁴. Heavy metal toxicity causes the oxidative stress which enhances the production of reactive oxygen species thereby decreasing the antioxidant systems (glutathione, superoxide dismutase, etc.) which protect cells⁵.

 

Due to high production cost, adverse effects, more chances of drug resistance of synthetic drugs, there is a boon in safe, potent, and economic herbal remedies⁶.  Herbal medicines are obtained from medicinal plant sources which include the dried herbs or fresh or dried part of the plant such as the leaves, roots, flowers, or seeds. Although herbal medicines are natural and considered to be harmless but their side-effects have been reported either due to their phytoconstituents or contaminants such as heavy metals, pesticides, microorganisms and adulterants etc⁷. The therapeutic efficacy of plants are due to the presence of active organic phytoconstituents like flavonoids, phenolic compounds, tannins, alkaloids, glycosides, essential oils, vitamins etc., but also there is a need to pay a little attention towards their inorganic constituents like heavy metals. Use of plants without estimating the concentration of trace elements (minerals and heavy metals) can lead to serious health problems⁸. Various official health organizations have set their own legal permissible limits of some heavy metals. For example, the Ministry of Health of Singapore stipulates that the legal permissible limits of As, Cu, Pb and Hg are 5, 150, 20 and 0.5 ppm respectively and World Health Organization have set the maximum permissible limits of toxic metals like As, Cd and Pb to an amount of 1.0, 0.3 and 10 ppm respectively for medicinal plants that has been used as raw materials for various herbal products^9,10.  Medicinal herbs are easily contaminated during growth, development and processing. Using herbs and their extracts for any therapeutic action, without estimating their heavy metal content may cause toxicity. Therefore, it is important to control the level of these contaminants in medicinal plants and detection of these metals in medicinal plants and their products should be done as a part of quality control in order to establish their purity, safety and efficacy¹¹.

 

P. betle L. also termed as Green Gold of nature as essential oil of leaves contains various valuable phenolic and terpenoids phytoconstituents such as carvacrol, eugenol, chavicol, allyl catechol, cineole, estragol, caryophyllene, cardinene, p-cymene and eugenol methyl ether etc¹². After tea and coffee, P. betle leaves (Paan) ranked second position based on daily consumption in India¹³. It has been used in various Ayurvedic formulations such as Lokantha Rasa, Puspadhava Rasa, Brhat sarwajwarahara lanha etc. Paan along with areca nut or tobacco has been offered as a symbol of respect to guests and elder persons¹⁴.  J. gossypifolia L. (Family: Euphorbiaceae) is commonly known bellyache bush or black physicnut. Decoctions of leaves have been used for bathing wounds. Due to valuable phytoconstituents such as alkaloids, amino acids, coumarins, steroids, flavonoids, lignans, proteins, saponins, tannins, and terpenoids etc. it possesses antimicrobial, analgesic, wound healing, antiulcer, haemostatic action¹⁵. Regarding the preceding comments, the main purpose of this research was to evaluate the content of heavy metals (Hg, As, Cd) and trace elements (Zn, Cu, Mg, Ca) in selected medicinal plants P. betle L. and Jatropha gossypifolia L. that has been traditionally used as alternative medicine in modern therapeutic system.

 

MATERIALS AND METHODS:

Chemicals:

All the chemicals used for present study were of high purity grade. Standard curves for calibration were prepared from respective salts of the metals. Ca as anhydrous calcium chloride (CaCl₂ ), Zn as zinc chloride (ZnCl₂), Cu as copper sulphate (CuSo₄), Mg as magnesium hydroxide (Mg(OH)₃) were purchased from Merck (Dormstadt, Germany). Standard solution of Cd, As and Hg of 1000 ppm were obtained from Spectrochem Pvt. Ltd. Mumbai.  HPLC grade deionised water was used in the present study.

 

Sample collection:

Fresh leaves of P. betle L. were collected from commercial sources from Jind and fresh leaves of J. gossypifolia L.  were obtained from Chandan vatika herbal garden, Jind District, Haryana (India).  The plant parts were identified and authenticated by taxonomist; Dr. Sunita Garg, Chief Scientist at CSIR-NISCAIR, Delhi (India) with reference no. NISCAIR/RHMD/Consult/2014/2466/45-2 and NISCAIR/RHMD/Consult/2014/2466/45-1 respectively. Voucher specimen of both plants has been deposited for future references.

 

Sample preparation:

The collected leaves were washed with distilled water and dried under shade for ten days. Dried leaves were ground to coarse powder using mechanical grinder and stored in an airtight container. The two test samples were analyzed by AAS (Electronics India Corporation Limited, AAS-4141) for the presence of Zn, Ca, Mg, Cu, Cd, As and Hg content.

 

Determination of metal content:

Wet digestion method:

Dried powdered leaves powder of P. betle L. and J. gossypifolia L. were digested by wet digestion method. Two grams of dried plant sample of P. betle L. was taken in a 100 ml beaker and 10 ml of concentrated nitric acid was added. The sample was heated at 95°C for 15 minutes on a hot plate. The digest was cooled and 5 ml of concentrated nitric acid was added and heated for additional 30 minutes at 95°C. The later step was repeated till the solution was reduced to about 5 ml without boiling. The digest was cooled again and 2 ml of water and 3 ml of 30% hydrogen peroxide was added to it. By covering the beaker, the sample was heated gently to start the peroxide reaction. If the excessively vigorous effervescence produced, sample was removed from the hot plate and 30% hydrogen peroxide was added in aliquots of 1 ml, followed by gentle heating until the effervescence was subsides. 5 ml of concentrated hydrochloric acid and 10 ml of water was added and the sample was heated for additional 15 minutes without boiling. At last, the sample was cooled and filtered through a whatman filter paper no. 42 (ashless) and diluted to 60 ml with HPLC grade deionised water¹⁶.

 

Preparation of calibration curve using standard solution:

Five serial dilutions of each element were prepared from their stock solution (1000 ppm) which is either available readymade or prepared from their respective salts with HPLC grade water depending upon the linear working range of each element.

 

Procedure for herbal sample metal analysis by AAS:

Atomic absorption spectrophotometer with flame atomization (air-acetylene) was used for determining the concentration of Cu, Zn, Mg, Ca, As, Cd, As and Hg metals in both samples.  The instrument was operated by personal computer using software Double-AAS. The samples and standards at different intervals were aspirated into flame by atomizer.  The heavy metals like As, Hg, Cd were converted into hydride form in acetylene-nitrous oxide flame using hydride generator. Maximum absorbance was obtained by adjusting the cathode lamps at specific slit width and definite wavelengths as shown in Table 1. Analysis of each metal was done in triplicates.

 

Data collection:

The concentrations of heavy metals were expressed as mean of heavy metal concentration (ppm) ± standard deviation (S.D) of triplicates. Calibration curve for each element was determined and regression coefficient was calculated. Concentrations of heavy metal in the samples were calculated from the calibration curve.

 

Data analysis:

The mean and S.D of each sample were calculated to analyze the unpaired student ‘t’ test value using Microsoft excel-2007 software. Significance of differences among the means was statistically analyzed (at P < 0.05).

 

RESULTS:

The results of the present study as shown in Table 2; revealed that the concentration of toxic elements i.e. As, Cd and Hg (below detecton limit) as well as other trace elements i.e. Cu (1.625 ppm), Zn (5.79 ppm), Mg (1027 ppm) and Ca (103 ppm) are within permissible limits in P. betle L. and the concentration of toxic elements i.e. As, Cd and Hg (below detecton limit) as well as other trace elements i.e. Cu (0.789 ppm), Zn (8.57 ppm), Mg (278 ppm) and Ca (427 ppm) are within permissible limits in J. gossypifolia L. which otherwise might be toxic at higher concentration. Magnesium is present as a chief trace element in P. betle L. sample and calcium is chief element of J. gossypifolia L.  sample as shown in Figure 1. The decreasing order of metal content present was observed as Mg > Ca > Zn > Cu > As > Cd = Hg and Ca > Mg > Zn > Cu > Cd = As = Hg in dried leaf samples of P. betle L. and J. gossypifolia L respectively. Heavy metals Cd, As and Hg were found to be below the detection limit in both the samples. All the data was statistically found to be significant when analyzed by student ‘t’ test having probability factor value less than 0.05 (P < 0.05).

 

Table 1: Different parameters for operating conditions of atomic absorption spectrophotometer

Parameters	Cu	Zn	Mg	Ca	Cd	As	Hg
Wavelength (nm)	324.7	213.9	285.2	422.7	228.8	193.7	253.6
Burner slit width (mm)	0.7	0.7	0.7	0.7	1	1	1
Cathode lamp  current (mA)	10	10	10	10	12	12	12
Gas pressure (pascal)	5	5	5	5	8-10	8-10	8-10

 

Table 2: Concentration of trace elements/heavy metals present in P. betle L. and J. gossypifolia L. dried leaves sample and their corresponding maximum permissible limit^{17, 18}

Trace elements/heavy metals	Maximum permissible limit (mg/kg)	Metal concentration in mg/kg on dried weight basis of sample
		P. betle L.	J. gossypifolia L.
Cu	150	1.625 ± 0.17	0.789 ± 0.87
Zn	150	5.79 ± 0.24	8.57 ± 0.76
Mg	2000	1027 ± 65.4	278 ± 34.87
Ca	-	103 ± 0.97	427 ± 0.117
Cd	0.3	bdl	bdl
As	10	bdl	bdl
Hg	0.5	bdl	bdl

bdl: below detection limit; Average value ± standard deviation here, n= 3, P < 0.05

 

Fig. 1. Concentration of trace elements in Piper betle L. and Jatropha gossypifolia L. dried leaf sample

 

DISCUSSION:

Poor quality control is one of the major hindrances in the global acceptance of herbal drugs. In case of herbal drugs, quality control of raw material is a big challenge since, they are of natural origin. The development of herbal drugs involve various processing steps like cultivation, collection, harvesting etc. which may itself act as a source of contamination¹⁹. Quality of herbal products directly affects their safety and efficacy.  However, quality control of herbal drugs by determining their identity, purity, phytocontent and biological property is a tedious and time consuming process. In addition to adulteration with spurious and misbranded substances contamination with undeclared toxic or hazardous substances are most likely to be found in herbal products. The toxic heavy metals, residual pesticides or improper use of sulfites are regarded as potent risk factors in use of herbal medicines²⁰. In recent years, it has been exposed by various researchers and health regulatory agencies that some traditional Chinese, Tibetan and Asian medicine systems contain significant amounts of Hg, As or Pb. It has been shown that herbal remedies incorporated in Asian traditional herbal medicine for therapeutic purposes caused intoxications in users. Therefore, medicinal plants which have been most commonly used in these traditional systems should meet the safety and regulatory guidelines of herbal medicine²¹. In the present research work, analysis of heavy metals in dried leaf sample of P. betle L. and J. gossypifolia L. has been done by atomic absorption spectrophotometry. Atomic absorption spectrometry (AAS) is a sensitive and most commonly used analytical technique for analyzing metals and metalloids. It is so sensitive that it can measure down to parts per billion of a gram in a sample. The technique makes use of the wavelengths of light specifically absorbed by an element. They correspond to the energies needed to promote electrons from one energy level to another, higher, energy level²².  P. betle L. belonging to family Piperaceae have been recognized as potential reservoir of phenolic compounds chiefly it contains eugenol and hydroxychavicol. It is highly demanded economic vine in South Asian countries for masticatory purpose as it possesses nutritive and digestive properties.  Paan leaves has been reported to show various medicinal properties such as antioxidant, antimicrobial, anti-inflammatory, anti-cancer activities due to the presence of number of bioactive compounds²³. Paan leaves has been traditionally used for chewing in their natural raw form along with other ingredients like sliced areca nut, slaked lime, coriander, aniseed, clove, cardamom, sweetener and flavouring agent etc²⁴. Jatropha gossypiifolia L. (Famlily: Euphorbiaceae) is commonly known as “bellyache bush” and is a multipurpose ethnomedicinal plant widely used in folk medicine for the treatment of various diseases due to antihypertensive, anti-inflammatory, analgesic, antipyretic, antimicrobial, antianemic, antidiabetic and antihemorrhagic activities [15].

 

CONCLUSION:

It may be concluded from present research work that the concentrations of the trace elements i.e. copper, zinc, magnesium and calcium in the dried leave powder sample of P. betle L. and J. gossypifolia L. were within the permissible limits, while the concentration of heavy metals As, Cd and Hg was below the detection limit. The studied medicinal plant samples were found to be safe for any kind of therapeutic application. Our findings also impose that the growing sites for these medicinal plants were appropriate and not contaminated with heavy metals. It is necessary to disperse awareness of such kind of phenomenon to prevent the collection of medicinal plants from non-cultivated and other sources such as land’s near sewerages and alongside industries which are highly contaminated with heavy metal contents. In addition, determination of heavy metal concentrations in medicinal plants used as raw material for formulating any kind of pharmaceutical, cosmaceutical, nutraceutical product must be taken as standard criterion for evaluation of their quality, safety, efficacy and purity.

 

CONFLICT OF INTEREST:

The authors do not have any conflicts of interest.

 

ACKNOWLEDGEMENTS:

The authors would like to acknowledge the financial support provided by University grant commission by providing UGC-BSR fellowship for research work. Authors would also like to thanks Department of Pharmaceutical Sciences, M.D. University, Rohtak (Haryana) for providing necessary laboratory facilities to carry out the research work.

 

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Accepted on 20.11.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 5078-5082.