Journal of Indian System of Medicine

: 2021  |  Volume : 9  |  Issue : 1  |  Page : 28--32

Experimental brine shrimp (Artemia Salina) lethality assay to evaluate the drug incompatibility on combined administration of Kakamachi (Solanum nigrum) and Madhu (honey)

Manjunath N Ajanal1, HN Sushma1, IB Kotturshetty2,  
1 Department of Dravyaguna, Rajiv Gandhi Education Society’s Ayurvedic Medical College, Hospital, Post Graduation Studies and Research Centre, Rona, Karnataka, India
2 Department of Panchakarma, Rajiv Gandhi Education Society’s Ayurvedic Medical College, Hospital, Post Graduation Studies and Research Centre, Rona, Karnataka, India

Correspondence Address:
Dr. Manjunath N Ajanal
Rajiv Gandhi Education Society’s Ayurvedic Medical College, Hospital, Post Graduation Studies and Research Centre, Ron, Karnataka.


Background: Drug incompatibility is one of the major causes of adverse drug reaction (ADR) or drug-induced toxicity in Ayurveda. In fact, this science has given special importance to such drug-induced issues and grouped them under the concept of Viruddha (drug incompatibility) and provided many measures to avoid them in clinical practice; one such example is a combination of fruits of Kakamachi (Solanum nigrum [SN]) and honey. The toxicity of this combination was not assessed; thus, the present study was planned to assess the toxic effect of the combined administration of fruits of Kakamachi and honey on brine shrimp lethality assay. Aims: To evaluate the brine shrimp lethality activity of the combined drug administration of fruits of Kakamachi and honey. Materials and Methods: Authentic brine shrimp eggs were laboratory cultured in artificially prepared saline sea water, and hatched brine shrimp nauplii were subjected to different concentrations of the combination to evaluate the LC50 and lethality assay. Results and conclusion: The study demonstrated that LC50 of fruit powder of SN and honey has shown potential cytotoxic effects at a dose of 0.57 mg on brine shrimp nauplii.

How to cite this article:
Ajanal MN, Sushma H N, Kotturshetty I B. Experimental brine shrimp (Artemia Salina) lethality assay to evaluate the drug incompatibility on combined administration of Kakamachi (Solanum nigrum) and Madhu (honey).J Indian Sys Medicine 2021;9:28-32

How to cite this URL:
Ajanal MN, Sushma H N, Kotturshetty I B. Experimental brine shrimp (Artemia Salina) lethality assay to evaluate the drug incompatibility on combined administration of Kakamachi (Solanum nigrum) and Madhu (honey). J Indian Sys Medicine [serial online] 2021 [cited 2021 Jun 14 ];9:28-32
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Ayurveda has been given prime importance on drug safety, and it is a fundamental concept in medical practice. Currently, many safety issues with respect to alternative medicine and Ayurveda are increased,[1],[2],[3] and this could be because of drug interaction, drug overdose, incompatibility, substandard drug etc.[4] Drug incompatibility is “The modification of the effect of one drug by the prior concomitant administration of another (precipitant drug)” cause of ADR, which caused harmful effects in any system of medicine.[5] Ayurveda explicates this issue under Viruddha (drug incompatibility); the combination of safe and harmful substance administration that produces some untoward effect is called Viruddha. The enormous explanation and restrictions while combining medicines are available in Ayurveda literature but scientific validation is yet to be generated by adopting newer experimental modules. One such incompatible combination is Kakamachi (SN) and Madhu (Honey).[6] In the current study, this combination was tested to evaluate the effect of the toxicity study (lethality) on brine shrimp.

The SN Linn belongs to the family Solanaceae, called as black night shade; the fruits are used in Ayurveda for various therapeutic modalities. The plant contains tannins, alkaloids, glycosides, flavonoids, saponins, proteins, carbohydrates, steroid alkaloid, coumarins, steroidal saponins, glycoprotein, and phytosterols. The fruit phytochemicals are solasodine, ascorbic acid, solanigrosides C–H, degalactotigonin, nigrumnins I and II, magnesium mineral etc.[7] This plant possesses toxicity due to the presence of solanine, which is a glycoalkaloid found in unripe berries. However, ripe fruit berries are least toxic. Pharmacologically, the plant exhibits antitumor, anticancer, antifungal, anti-larvicidal, anti-stress, antioxidative, anti-allergic, estrogenic, hepatoprotective, anticonvulsant, antidiabetic, immunostimulant, antimicrobial, cardioprotective, analgesic, antidiarrheal, cytotoxic, anti-inflammatory, anti-seizure activity etc.[8]

Honey is a product of honey bees; it is known for its good therapeutic effects. It contains many therapeutically beneficial substances, mainly fructose, fructo-oligosaccharides, glucose, vitamins, minerals, amino acids, and enzymes. In Ayurveda, this has been used as an adjuvant in many therapies. Its pharmacological activities are bactericidal, anti-inflammatory, immuno-stimulant, cardio-protective, and antineoplastic in nature. No toxicity reports exist on its uses.[9]

The combination of Kakamachi and Madhu (Honey) is considered as one of the incompatible combinations explained in Ayurveda.[6] Phytochemicals such as dillapiole, α-cadinol, paracymene, αphellandrine etc. of Kakamachi and amino acids and proteins of honey may interact with each other and produce toxicity. Similar to other studies, this study showed the toxic effects of a combination of honey and ghee.[10] Thus, a validation of this procedure is undertaken to study the brine shrimp lethality assay.

The selected assay is capable of killing laboratory-cultured brine shrimp nauplii (Artemia salina).[11],[12],[13] The method is one of the simplest and accurate tools to test plant extract toxicity. The assay was also used extensively to assess plant toxicity due to heavy metals, detection of aflatoxins, pesticides, cytotoxicity etc.[14],[15] The brine shrimp assay method is the most common method that is used to assess the toxicity because of its simplest technique, low cost, and sensitivity. In the present work, the cytotoxic effect of the combined administration of Kakamachi (Solanum nigrum) and Madhu (Honey) is reported.


Collection of Materials

Kakamachi fruits were collected from Kappathagudda forest area, and honey was purchased from an authorized vendor. Both drugs were authenticated and confirmed by preparing a voucher specimen along with herbarium specimens at Central Research Centre (CRC) of RGES AMC Ron, with RGES/AMC, HandPG/CRC/Aut/2020–99 as authentication no. Thus, the confirmed fruits of SN were further dried under shade and stored [Figure 1]. The collected and authenticated honey was used for further evaluation. Quality assessment of fruits was carried out according to API.[16] A TLC study of alcoholic and water extract of fruits was plotted on silica gel “G” plate using the mobile phase of Toluene: Ethylacetate (90:10).[16]{Figure 1}

Preparation of Drug Dosage Form

The drug dosage form was prepared with an equal volume (W/W) of fruit powder, fruit powder water extract, and fruit powder alcoholic extract with honey in different concentrations from 0.1 mg, 1 mg, 5 mg, and 10 mg; these were administered along with individual drugs and standard control drugs such as podophyllotoxin.

Cytotoxicity Bioassay

Brine shrimp cytotoxicity assay was carried out to investigate the ability of killing nauplii by the administration of combined Kakamachi fruits and Madhu.[11],[12],[13]

Collection of Brine Shrimp Eggs

Brine shrimp eggs were purchased from authorized suppliers and were used for further analysis.

Preparation of Artificial Sea Water

One liter of artificial sea water was prepared by dissolving 38 g of sea salt (NaCl) in one liter of distilled water, and pH was adjusted to 8.5 by using 1N NaOH solution.

Brine Shrimp Hatching

A 1-liter-capacity conical hatching chamber was selected; half of its portion was covered with black paper, and the other half was without any cover. The artificially prepared sea water was added to a conical flask along with eggs of brine shrimps (Artemia salina), and the flask was constantly aerated for 48h. After 48h, the hatched brine shrimps were accumulated at the brighter side of the flask and they were used for further cytotoxic activity.

Grouping and Experimental Study

The active nauplii free from egg shells from the brighter portion of the chamber were collected and used for the assay. A total of nine groups were prepared and served as controls: no drugs, fruit powder, honey, fruit water extract, fruit ethanol extract, fruit powder + honey, fruit water extract + honey, fruit ethanol extract + honey, and positive control (Podophyllotoxin) with different concentrations of 0.1 mg, 1 mg, 5 mg, and 10 mg, respectively.

Through a glass capillary ten nauplii were drawn and placed in each vial containing 4.5mL of sea water and 0.5mL of the prepared dosage form drug solution. The vials were kept in a controlled environment with a temperature of 25°C and light for 24h, and the mean surviving larvae were counted. The same procedure was conducted along with control (vehicle treated), different concentrations (0.1–10 mg/mL) of the test substances in a set of three tubes per dose. Podophyllotoxin was used as a positive control in the assay [Figure 2].{Figure 2}

Lethality Concentration Determination

The estimation of lethality percentage was done by comparing with test samples mean surviving larvae and control. The obtained values were further plotted on a graph to obtain an LC50 value by the best-fit line plotted concentration versus lethality percentage.


Organoleptic Study

The fruit berries were broadly ovoid, purple to blackish or yellowish-green with 5–9mm broad; these were sweetish to bitter in taste and had a sweet odor. Each fruit contained 1.7–2.5mm long-sized seeds that were 24 to 60 in number [Figure 1].

Powder Study

The powder was smooth, fine, dark brown in color, had a faint odor, and was bitter in taste.

Fruit Microscopy

The fruit had an epicarp and was very thin and papery, with a pulpy mesocarp and exile placentation; the seeds were freely suspended in pulp. Powder microscopy showed the presence of phloem fibers, fragments of pericarp, fibers, stone cells, starch grains etc.

The fruit powder physicochemical analysis showed 9% of ash value, 4% as acid insoluble ash, 6% as water-soluble ash, 20% as water extractive value, 12% as alcoholic extractive value, 4% as moisture content, and 1% of foreign matter, respectively. The fluoroscence and behavioral study is presented in [Table 1], the preliminary phytochemical study is presented in [Table 2], and the TLC study is presented in [Table 3].{Table 1} {Table 2} {Table 3}

Cytotoxic Study

The study demonstrated that brine shrimp lethality was interrelated with a concentration of the combination of honey and SN; toxicity was increased with an increase in concentration. This was estimated in the form of LC50 by plotting on a graph.

The LC50 values of the brine shrimp obtained for the combined administration of fruit powder and extract with honey along with individual powder, extracts, and positive control (podophyllotoxin) are shown in [Table 4]. The combined fruit powder with honey, water extract with honey, alcoholic extract with honey, plain powder, plain water extract, alcoholic extract, and podophyllotoxin is indicated as 0.57 mg, 0.33 mg, <0.1 mg, 5 mg, <5 mg, <0.5 mg, and 1.43 mg, respectively [Figure 3].{Table 4} {Figure 3}


The outcomes of the analytical parameters of fruits of SN were within the standard reference values; this indicated that the drug was genuine.[16],[17]

The current chosen method for the evaluation of the effect of drug toxicity on the brine shrimp model is that it is an easy and cost-effective experiment and can be performed in a small setup; thus, this method can be made mandatory for the gross assessment of drug toxicity.

The study showed that cytotoxicity was very prominent in brine shrimp that is 0.57 mg, which is more even compared with a positive control (podophyllotoxin) of 1.43 mg; however, the individual drugs were not shown to exhibit prominent toxic effects compared with the control group. This shows that the Ayurveda concept of Virudha (incompatibility) with respect to Kakamachi and honey is hazardous and, hence, this combination is contraindicated in clinical practice. The cytotoxic effect was more from water extract (0.33 mg) than alcoholic extract (<0.1 mg); this could be due to the highest proximity of the polar solvent nature of alcohol.

The present positivity of toxicity on brine shrimp by the administration of this combination could be due to the presence of traces of phytochemicals of SN, such as solanaceous glycoalkaloids, which are prominent known toxic chemicals. Although this chemical is present in traces, on combination with honey it may interact with it and accelerate the toxic effect.[18]

The present study was maiden in its nature to the Ayurveda system, to test the toxicity of Viruddha drugs and it provided baseline information about toxicity. The actual mechanism of toxicity by adopting the brine shrimp assay is very difficult to understand but it created a hypothesis that there is strong positivity between Ayurveda drug incompatibility and toxicity; further, these data can be extensively studied in higher animals so as to understand them.


The study suggested that the Ayurveda concept of Viruddha (incompatibility) of a combination of fruit powder of SN and honey has shown potential cytotoxic effects at LC50 0.57 mg on the brine shrimp lethality assay.


The authors acknowledge RGUHS Bangalore for providing financial support in conducting this study and Vision Group on Science and Technology (VGST) Govt. of Karnataka for up-gradation of the research analytical lab, which enabled the authors to conduct this research smoothly.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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