1.1. Taxonomic Status

Kingdom: Plantae, Class: Magnoliopsida, Order: Lamiales, Family: Acanthaceae, Genus: Justicia, Species: adhatoda [25].

In Unani and Ayurvedic medicine, J. adhatoda is a well-known plant medicine [26]. It is an Ayurvedic herb used to treat coughs, asthma, bronchitis, and common colds [24]. It is the source of medication “Vasaka” which is widely recognized in traditional medicine for its curative properties, especially in the treatment of bronchitis [27]. Antispasmodic, anti-inflammatory, bronchodilator, antibleeding, disinfectant, antijaundice, antidiabetic, fever reducer, and oxytocic are some of the worth mentioning medical properties of this plant [28]. This plant has astringent, diuretic, purgative, antiperiodic, and expectorant effects and is capable of liquefying sputum [29]. Its leaves, flowers, and roots are applied in herbal medicines to treat, cancer [30] and tuberculosis [31], and exhibit antihelmintic effects [32]. The leaf extract is reported to be useful in treating dysentery, diarrhea, and glandular tumours [32].

J. adhatoda has been extensively researched for its phytochemical and pharmacological properties. It is included in the class of herbal medicines that have a very strong traditional basis. J. adhatoda contains significant amounts of vasicine, vasicolone, vasicinone, and other alkaloids. The plants has antibacterial, antifungal, antitussive, anti-inflammatory, hepatoprotective, antiulcer, antiviral, abortifacient, thrombolytic, antimutagenic, hypoglycemic, and antioxidant properties [33]. After performing phytochemical evaluations on various parts of J. adhatoda, namely on bark, fruit, flower, root, wood, and even the whole plant, approximately 233 phytochemicals have been identified so far. Among them are 12 flavonoids, 33 alkaloids, 47 essential oils, 47 organic acids, 23 terpenes and steroids, 14 phenolic compounds, hydrocarbons including alkanes, alkenes, alkylamine, acetylene, naphthalene, naphthoquinone, fatty alcohol, and 59 miscellaneous chemicals [34]. Additionally, some macro and micro minerals have been found in plant, such as calcium (Ca), chromium (Cr), copper (Cu), iron (Fe), potassium (K), manganese (Mn), vanadium (V), and zinc (Zn). [35].

2.1. Sample Preparation and Experimental Details

Plant leaves were collected in September 2021 from the Soon Valley (32°58′N 72°15′E) and Chinji National Park (33°0′36.87″N 72°29′30.98″E), Punjab, Pakistan. The leaves were cleaned, dried (at room temperature), ground, and converted into a fine powder. In order to obtain a pure extract of the plant, 50 g of leaf powder was mixed with 500 ml of methanol. A pure extract of the plant was achieved after filtration and evaporation. The extract was weighed after being concentrated, labeled, and kept in small sterilized bottles at 4°C for further analysis.

2.2. Phytotoxic Effect

The phytotoxic activity of J. adhatoda was analyzed with the help of root length inhibition and percentage inhibition in seed germination assays [36, 37]. Five different concentrations (0, 10, 100, 500, and 1000 ppm) of methanol extract were used in this experiment. To execute these assays, sterilized seeds were placed in petri dishes lined with two filter papers, and 5 ml of each solution was added to each petri dish.

2.3. Determination of Root Length Inhibition

2.4. Determination of Germination Percentage

One hundred seeds of radish and 70% methanol extract with five different concentrations (0, 10, 100, 500, and 1000 ppm) were used in this experiment. The germination rate of seeds was noted for five days. The percentage of germination was calculated by dividing the number of germinated seeds by the total number of seeds sown in petri dishes and multiplying by 100, as described by Bhardwaj [38]. Experiment was performed three times to calculate the mean values and standard deviation for all treatments.

2.5. Cytotoxic Effect

2.6. Proximate Composition Analysis

Proximate analysis was carried out to determine the percentage of moisture content, protein, dry matter, ash content, fat, and crude fiber. Moisture content was quantified using Nancy Trautmann’s method. The well-known Kjeldahl method was used to determine the crude protein percentage also described by Onwuka [40]. Fat extraction was performed using a Soxhlet apparatus. Acid-base treatments were used to estimate the crude fiber percentage. Dry matter was calculated using a protocol outlined in AOAC [41].

4.1. Seed Germination Percentage

Results illustrate the allelopathic/phytotoxic effects of J. adhatoda extract on radish seed germination. Seed germination decreased by increasing the concentration of extract. Germination percentage decreases up to 62.33. Means of seed germination percentage are 93.67%, 85.33%, 81.00%, 75.67%, and 62.67% in 0 ppm, 10 ppm, 100 ppm, 500 ppm, and 1000 ppm extract, respectively (Figure 1 and Table 1).

Table 2 reveals the ANOVA on the effect of J. adhatoda extract on germination of radish seeds. A substantial difference was observed between the treated and control sample.

Similarly, Khan et al. assessed the phytotoxic activity of J. adhatoda against duckweed (Lemna minor L). They tested different fractions like crude methanolic extract, n-hexane, CHCl_3, ethyl acetate, and aqueous fraction [43]. These fractions proved to be phytotoxic and are consistent with our findings.

In a same way, Devkota et al. [44–46] studied that J. adhatoda and Costus speciosus had the highest inhibitory effects on seedling growth and germination of wheat, pea, and turnip, respectively. Furthermore, they observed that J. adhatoda plant extract potentially impaired anabolic activities in plants along with the visible inhibition on seed germination parameters [46, 47]. Our results are in harmony with previous studies conducted on phytotoxic effect of J. adhatoda. We have observed significant inhibitory effects on seedling growth in radish seed.

4.2. Root Length Inhibition

The effect of five concentrations (0, 10, 100, 500, and 1000 ppm) of J. adhatoda methanolic extracts on root length indicated that root inhibition increased by increasing concentration of extract (Tables 3 and 4, Figure 2). Root length of radish seedling decreased up to 83.11%. Means of root inhibition are 24.00%, 47.11%, 62.22%, and 83.11% in 10 ppm, 100 ppm, 500 ppm, and 1000 ppm extract, respectively (Figure 2).

Table 4 reveals the analysis of variance of effects of J. adhatoda extracts on root inhibition of radish seedlings. A significant difference between control and treated samples was observed.

Allelochemicals present in the plant extract might be responsible for inhibiting root growth in the plants being studied. These types of growth inhibition by allelopathic plants extract have also been reported by Islam et al. [48, 49].

They observed that the allelopathic activity of Leucas aspera is due to the presence of growth inhibitory substances. According to Devkota and Sharma, aqueous extracts of J. adhatoda and Costus speciosus plants slightly inhibited pea root and hypocotyl, at a 2% concentration. The inhibitory effects increased when the concentration was increased to 10%. Wheat root length decreased from 5.78 ± 1.33 cm in the control to 0.84 ± 1.96 cm at a 10% concentration of J. adhatoda leaves [44].

Shinwari et al. evaluated the allelopathic effects of 160 medicinal plants collected from Tanegashima, Japan. Melia azedarach, Tylophora tanakae, and Cinchona sp. have inhibitory potentials ranging from 80 to 100% against lettuce root development, while Justicia procumbens and 9 other plants show root inhibition between 60 and 79% [50]. These results are in agreement with the phytotoxic effect of J. adhatoda extract in our study. Gautam et al. investigated the allelopathic potential of six plants on radicle length of maize seeds. Among the six plants, Parthenium hysterophorus extract showed the maximum reduction in radicle length of maize seedlings and a mild concentration of Lantana camara extract induced the maximum reduction in plumule length [51]. Similar results were also found by Devi and Dutta that 10% concentration of P. hysterophorus foliar extract causes a high level of radicle growth inhibition of maize [52]. Our findings are in accordance with these previous studies which were planned to assess root length inhibition by allelopathic chemicals of different plants. Growth parameters (root and shoot length, seed germination) of other plants are inhibited by allelochemicals. Allelochemicals may change membrane permeability, inhibit nutrient uptake, inhibit cell division and elongation, change submicroscopic structure of cell, affect plant photosynthesis and respiration, change various enzyme functions, and also affect the synthesis of plant endogenous hormones and proteins [53].

4.3. Cytotoxicity Assessment of Justicia adhatoda Leaves Extract

The cytotoxic effect was studied by brine shrimp lethality assays. Concentrations of 10 ppm, 100 ppm, and 1000 ppm were tested to determine the mortality in percentage (%) of brine shrimp nauplii. The mortality was 10%, 36.67%, and 76.67%, respectively, for J. adhatoda at these concentrations (Table 5, Figure 3).

In investigation, the LC50 of J. adhatoda extract was 217 µg/ml, which is considered to be cytotoxic.

Similar findings were also reported by Khan et al. [53]. They found that crude methanolic extract of J. adhatoda exhibited 13.33%, 3.33%, and 0% cytotoxicity at 1000, 100, and 10 g/ml, respectively [53]. Krishnaraju et al. revealed the results of screening of some medicinal plants for Brine shrimp cytotoxicity, in which Aristolochia indica, Boswellia serrata, Garcinia cambogia, Ginkgo biloba, and Semecarpus anacardium were shown to be considerably cytotoxic [54]. It was observed by Meskat and Hussain that the LC50 values of ethyl acetate, chloroform, and n-hexane-soluble fraction were found to be 1.402, 2.130, and 1.129 µg/ml, respectively [55]. In a same way, Patel and Zaveri observed that the methanolic extract of Justicia gendarussa root and leaf showed a notable cytotoxic effect on brine shrimps. Active fraction of 100 μg/ml dose level elicited 100% hatching inhibition and showed an LD50 value of 25.44 μg/ml in the toxicity assay, which might indicate cytotoxic activity. The LD50 of the isolated compound from the toluene fraction was 8.13 g/ml [56]. Sadek investigated the impact of crude methanolic extracts of Adhatoda vasica leaves on the feeding of Spodoptera littoralis larvae. Feeding on fresh leaves caused 100% mortality of larvae after 26 days of insubstantial growth. The extract showed strong toxic and antifeedant activity against the larvae [57].

The cytotoxicity of gandarusa (Justicia gendarussa Burm.f.) was studied by Widodo et al. by Brine Shrimp Lethality Test (BSLT). They reported LC₅₀ of 96% water extract and ethanol extract of J. gendarussa leaves to be 18.02 μg/ml and 713.34 μg/ml, respectively [58]. In mice, the hydro-alcoholic extract of Justicia vahlii has been found to be nontoxic up to 4000 mg/kg, and brine shrimp lethality assay showed no mortality [59]. We have observed significant cytotoxicity of 76.66% of J. adhatoda extract against brine shrimp at 1000 ppm concentration.

4.4. Proximates Analysis of Justicia adhatoda Leaves

Proximate analysis revealed that leaves of J. adhatoda have 9.4% moisture, 90.6% dry matter, 19.25% crude protein, 4.5% crude fat, 8.0% crude fiber, and 11.5% total ash (Table 6).

Similar to our findings, Baniya reported that the moisture content of leaves is 10.2% [60]. Our findings digress from those of Jayapriya and Shoba, according to whom the moisture content of J. adhatoda leaves was 18.20% [61]. Gulfraz et al. reported that Adhatoda vasica leaves had a 15.3% moisture content while roots had 24.6% [62]. According to Kumar et al., the moisture content of J. adhatoda leaves was 15.3 ± 0.5% [63]. Our findings revealed that plant leaf powder contains 90.6% dry matter, while according to Gulfraz et al., J. adhatoda have 50.4% and 66.4% dry matter in leaves and roots, respectively [62], which deviate from our findings. The dry matter content of J. secunda was also reported by Arogbodo to be 87.20 ± 0.25% [64].

The percentage of crude proteins in dried leaves of J. adhatoda is 19.25%. It is in conformity with Rasheed et al. [65]. They reported that J. adhatoda leaves had 21.33% protein content, which is almost near to our findings [65]. Gulfraz et al. reported that protein content was 8.5% in roots and 6.5% in leaves of J. adhatoda [62]. According to Kumar et al., the protein content of J. adhatoda leaves was 6.5 ± 0.3% [63]. The crude protein content of J. secunda was reported by Arogbodo to be 18.09 ± 0.18% [64]. Ajuru et al. showed that the leaves of J. secunda contain (22.33 + 0.02%) protein content, which was greater than the present findings of J. adhatoda protein content, and the leaves of J. carnea had a protein content of 17.53 + 0.02% [66]. This is slightly lower than our findings.

In our findings, crude fat percentage in dried leaves of J. adhatoda is 4.5%. Gulfraz et al. reported that fat content was 2.5% in roots and 1.6% in leaves of J. adhatoda [62]. According to Kumar et al., the crude fat of J. adhatoda leaves was 1.6 ± 0.3%. Our findings showed deviation from these findings [63]. Arogbodo reported that crude fat of J. secunda was 8.10 ± 0.39% [64].

Crude fiber percentage in dried leaves of J. adhatoda is 8.0. According to Kumar et al., the ash content of J. adhatoda leaves was 6.4 ± 0.45%, which is in harmony to our findings [63]. Arogbodo reported that crude fiber of J. secunda was 0.60 ± 0.14% [64]. Ajuru et al. reported that fiber content values of J. carnea and J. secunda were 42.53 + 0.00 and 42.21 + 0.02%, respectively, which were much greater than the present findings [66]. The percentage of total ash in dried leaves of J. adhatoda is 11.5%. Baniya reported similar findings, indicating that the total ash value was 13.3% [60]. Palshikar and Pandiyan reported that the ash content was 9.5% in J. adhatoda leaves of rainy season [67]. According to Gupta et al., physiochemical studies of J. adhatoda revealed that the plant contains 20% total ash, 82% acid-insoluble ash, and 4.5% water-soluble ash [68]. Jayapriya and Shoba narrated that the total ash content, water-soluble ash, and acid-insoluble ash of J. adhatoda leaves were not more than 21.40%, 4.85%, and 0.92%, respectively [61]. The ash content in J. secunda and J. carnea was reported by Ajuru et al., which was 15.62 + 0.03 and 15.01 + 0.01%, respectively [66]. Reddy et al. reported that the total ash content of J. adhatoda flowers was 6.89% [69].

4.5. Qualitative Analysis of Phytochemicals

Plant extracts were scrutinized for the determination of different phytochemicals. Different phytochemical tests were performed to detect the presence of various phytochemicals. The results of phytochemical analysis of J. adhatoda leaves extract are shown in Table 7, which indicates the presence of various phytochemicals, such as alkaloids, coumarins, flavonoids, phenols, saponins, and tannins (Figure 4).

The phytochemical assessment was carried out on the crude methanolic extracts of J. adhatoda leaves. Our findings showed the presence of alkaloids, coumarins, flavonoids, phenols, saponins, and tannins. Similarly, Baniya indicated the presence of alkaloids, carbohydrates, cardiac glycoside, flavonoids, reducing sugar groups, steroids, saponins, tannins, and vitamin C in J. adhatoda leaves [60].

According to a group of Indian researchers, major chemical elements of Adhatoda vasica leaves include pyrroloquinazoline alkaloids, adhatonine, vasicine, vasicinone, vasicol, vascinolone, and vasicinol. Vasicine has been shown to have bronchodilatory, uterine, and respiratory stimulant properties. Vasicinone was also shown to have bronchodilatory and antianaphylactic action and weak cardiac stimulant [70].

The alkaloids have antioxidant, antidiabetic, antimicrobial, anti-inflammatory, antiallergic, abortifacient, uterine stimulant, bronchodilatory, and electrophoretic effects [71–73]. Vinothapooshan and Sundar reported that various leaf extracts of the plant Adhatoda vasica contain biologically active phytochemicals such as alkaloids, essential oil, flavonoids, quinazoline, tannins, and vasicinone, which are accountable for the significant hepatoprotective activity [74]. The flowers also contain a very good amount of phenolic compounds and flavonoid contents [75].

According to Chaudhary et al., phytochemical analysis of Adhatoda vasica leaves in methanolic extract, ethyl acetate, and aqueous extract exhibits the presence of saponins, carbohydrates, flavonoids, tannins, and alkaloids [76]. Similarly, according to Sarker et al., the ethanolic, petroleum ether, and water extracts of Adhatoda leaves are enriched with alkaloids, phenols, tannins, and reducing sugars. Main constituents like vasicine and others have been found to possess mild antibacterial activity against some microbes [77]. Vinothapooshan and Sundar suggested, according to their experimental data, that bioactive phytochemicals such as alkaloids, essential oil, flavonoids, quinazoline, tannins, and vasicinone present in the several extracts of the Adhatoda vasica plant may be responsible for the potential hepatoprotective activity [74].

Francis et al. demonstrated in experimental animals that saponins present in Justicia plants have antiviral, antifungal, antiprotozoal, and cytostatic activities on various types of cancer cells, cell-mediated immune system stimulation, lower serum cholesterol, and antibody production enhancement [78]. Tannins have strong antibacterial, antifungal, and antiviral properties as well as biological activity relating to their potential for protein precipitation and astringency, which have led its use as an antidiarrheal, wound healer, and antiseptic [79]. On the other hand, several coumarins have shown biological actions such as anti-inflammatory, anticoagulant, antioxidant, and analgesic activities [80]. The presence of these compounds assumes excellent pharmacological potential for Justicia species.