Study Area

The study area, the Asir region (Figure 3), is located in the southwestern part of Saudi Arabia between latitudes 17°25′ and 19° 50′ North and longitude 50°00′ and 41°50′ East (El-Juhany & Aref, 2013). The region is situated on a high plateau of an area approximately 81,000 km², with the elevation ranging from 1,000 m to 3,130 m above the sea level. It contains mountains, slopes, rolling lands, rocky hills, deep valleys, and wadis. It is characterized by high rates of rainfall and moderate temperatures throughout the year (Seraj et al., 2014). The textures of the mountain soils of the region range from loamy silt to silty clay to sandy loam with a high percentage of organic matter, reflecting the favorable climatic environment for plant development. Such topography, along with a relatively mild climate, creates a great diversity of plant communities and habitats for various plants species (Al-Qahtani, 2000; Aref, 1996), such as Acacia species, Juniper trees, Ziziphus spina Christi, T. aphylla, Dobera glabra, Adenium obesum, Mimusops laurifolia, Ficus sycomorus, Tamarindus indica, and many others herbs and shrubs (Masood & Asiry, 2012). The population of the region is estimated to be 2,212 million (The General Authority for Statistics, 2017). The capital of Asir region is Abha. In addition, the region contains some main cities, including Khamis Mushait, Bisha and Bareg, and their surrounding suburbs.

Figure 3.

Map of the Saudi Arabia showing its 13 regions and the Asir region (in green) with its two main cities where the survey was carried out. Abha is located at 18° 13′ 0.4692″ N latitude and 42° 30′ 13.5540″ E longitudes, and it is situated at an elevation of 2,228 m above the sea level. Khamis Mushait is located at 18° 19′ 45.7824″ N latitude and 42° 45′ 33.7140″ E longitudes, and it is situated at an elevation of 1,998 m above the sea level. This image is prepared with the help of an editable map of Saudi Arabia (Adapted from  https://yourfreetemplates.com).

Botanical Identification

Several ethnobotanical field trips were carried out in the area around Abha and Khamis mushait cities during September/December 2018 to collect voucher specimens and photograph the selected plants. The plant’s species were authenticated using The Plant List (2013) and the available relevant scientific publications of Saudi Arabia Flora (Chaudhary, 1999, 2001). Further confirmation was made with the assistance of Dr. E. Warag, Professor, Department of Biology, King Khalid University, Saudi Arabia. Voucher specimens were processed using standard taxonomic procedures (Bridson & Foreman, 1998) and were deposited for future reference at the Herbarium of Biology Department, College of Science, King Khalid University.

Ethnobotanical Data Collection and Literature Review

Ethnobotanical information on the selected plants was obtained using semistructured interviews with local people (older age), traditional medicine practitioners (Hakim), and local herbal drug sellers (Atar) who live or work in Abha and Khamis Mushait cities. Study participants were asked (in Arabic) about their knowledge of the three selected plants using the local name of the plant and the plant images. Specifically, other local names (if any) of each species, the medicinal uses, the diseases commonly treated with the plant, the plant part(s) used, and the modes of preparation were recorded.

A systematic literature review using different databases such as Google search, Google Scholar, and the Saudi Digital Library was conducted to collect and assess the documented information about the selected plants with a focus on the Saudi species. The literature review surveyed all the available documents and covered medicinal uses, biological and pharmacological activities, and chemical constituents of the selected plant species using the search terms “Tamarix aphylla,” “Aerva javanica,” “Medicinal Plant,” “Traditional Medicine,” “Ethnobotany,” “Saudi Arabia,” and “Asir region” independently or in combination.

Use Value

The use value (UV) index (Andrade-Cetto & Heinrich, 2011) was used to evaluate the relative importance of the plant species based on its relative use among informants, according to Equation 1:

(1)

Plant Samples and Extract Preparation

After confirming plant identity with informants, the plant samples including the bark of T. aphylla and roots and aerial parts of A. javanica were collected from three locations in the study area. The plant materials were cleaned, air dried at room temperature, and powdered with a mechanical grinder. Each plant sample (250 g) was macerated in ethanol (80% v/v) at room temperature with occasional stirring. Each extraction was repeated 3 times, and the collected filtrates were combined and concentrated under reduced pressure using a rotary evaporator (RV 10, IKA®-Werke GmbH & Co. KG, Staufen, Germany). The obtained dried extracts were weighed to determine the percentage yield and then were stored in a refrigerator (4°C) until they were used for analyses.

Phytochemical Screening

Qualitative phytochemical screening was subjected to each plant sample extract to detect the different classes of phytochemicals present by adopting standard protocols (Harborne, 1998; Trease & Evans, 1989). The results were qualitatively expressed as the relative abundance.

Estimation of the Total Phenolic Content

The total phenolic content of plant extracts was estimated using the Folin-Ciocalteu spectrophotometric method as previously described by Nabavi, Ebrahimzadeh, Nabavi, Hamidinia, and Bekhradnia (2008), with minor modifications. The absorbance was measured at 760 nm using a JASCO V-530 UV/Vis spectrophotometer. Gallic acid (Sigma-Aldrich) was used as a standard to prepare the calibration curve. The total phenolic value in all samples was calculated and expressed as milligrams of gallic acid equivalents per 100 g of dry weight of the plant extract (mg GAE/100 g).

Estimation of the Total Flavonoid Content

The total flavonoid content of different plant extracts was estimated spectrophotometrically by the aluminum chloride assay described by Ordoñez, Gomez, Vattuone, and lsla (2006), with slight modifications. The absorbance of the treated sample and standard quercetin (Sigma-Aldrich) solutions were measured at 415 nm. Quercetin standards were used to produce the standard curve. The total flavonoid values in the test samples were calculated, and the results were expressed as milligrams of quercetin equivalents per 100 g of the dry weight of plant extracts (mg QE/100 g).

Evaluation of Antioxidant Activity

The antioxidant activity of the plant extracts was estimated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay as previously described by Eshwarappa, Iyer, Subbaramaiah, Richard, and Dhanajaya (2014) with minor modifications. The absorbance of the plant extract and standard solutions of ascorbic acid (positive control) were recorded at 517 nm. The percentage of inhibition of DPPH free radicals was calculated using Equation 2:

(2)

The hydrogen peroxide (H₂O₂) scavenging assay, as reported by Ruch, Cheng, and Klaunig (1989), was also applied to estimate of the antioxidant activity of the plant extracts. The absorbance of the reaction mixture was recorded at 230 nm, and the H₂O₂ scavenging effects of both the extract and standard were calculated as percentages using Equation 2. The scavenging percentages calculated for the different concentrations of plant extracts were used to calculate the IC₅₀ values using regression analysis.

Evaluation of Antimicrobial Activity

The plant extracts were screened for their activities against some human pathogens: Gram-positive (Candida albicans and Staphylococcus aureus) and Gram-negative (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Shigella flexneri) obtained from the Biology Department, King Khalid University. The pathogens were inoculated onto the nutrient agar plate and were incubated at 30°C for 48 h. The test was performed by the disc diffusion method (Berghe & Vlietinck, 1991; Ruangpan & Tendencia, 2004). The antimicrobial activity was calculated as the average zone (in mm) of pathogen growth inhibition. Ampicillin and nystatin (100 µg/mL) were used as positive controls for antibacterial and antifungal activity, respectively.

Statistical Analysis

Experiments were carried out in triplicate, and the data for each sample were recorded as means ± standard deviation (n = 3). MS Excel and SPSS (version 21) were used to perform statistical analysis. For all the analyses, the differences were considered to be significant at p ≤ .05.

Medicinal Values of the Plants

Table 1 represents the medicinal uses mentioned by the informants for each species. For each cited folk species, the scientific name, family, voucher number, local folk name(s), growth habit, part/medicinal uses, and UV were reported. Sixty-one selected informants, including 50 older men (50 to 72 years), 4 traditional medicine practitioners (Hakim), and 7 herbal drug sellers (Atar), were interviewed. The respondents confirmed more than one local name for each species. They also indicated that the best time to harvest the plants from the wild is in the spring before or during flowering. The ethnobotanical survey results revealed that the two plants could treat 18 types of diseases and disorders. Almost all the respondents knew about these species and confirmed that they were commonly used by the local communities to treat various human ailments. Most of the respondents reported that they had used these plants at least 1 time and that they advise neighbors and friends to use the plants. The typical plant parts mentioned by the study participants included leaves, bark, root, and aerial parts, while the preparation methods used included decoction, infusion, maceration, juice, powder, and paste. The most frequently reported ailments were rheumatism, wounds, skin diseases, pain, and kidney problems. The results showed that the same part was used to treat different conditions. For example, the leaves of T. aphylla were used to treat fever, wounds, jaundice, and rheumatism, and the roots of A. javanica were used to treat toothache, headache, kidney stones, and diarrhea. Generally, the UVs for the three plants were high (Table 1). A. javanica was the most frequently used species in the local area according to its high UV (0.9). It was mentioned by 90% of all the informants. T. aphylla reflected a UV of 0.7. The literature search results are presented in Table 2 with the relevant references. The results revealed that different species of T. aphylla and A. javanica from other countries were previously studied for chemical constituents and biological and pharmacological activities. Various phytochemicals, such as tannins, alkaloids, saponins, glycosides, and flavonoids, were recorded for each of the two species along with different isolated phytochemical compounds.

Table 1.

List of Indigenous Selected Plants With Their Families, Local Names, Growth Habit, and Traditional Uses Recorded in the Study Area With Their UVs.

Table 2.

Documented Medicinal Uses, Biological Activities, and Phytochemicals of the Study Species.

Extraction Yield and Phytochemical Profiling

The extraction yields of ethanol extracts of the bark of T. aphylla and roots and aerial parts of A. javanica were 12.3%, 5%, and 6.7%, respectively. The results of preliminary phytochemical screening, represented in Figure 4, revealed the presence of alkaloids, glycosides, saponins, triterpenes, tannins, and flavonoids with different abundance levels in the three plants extracts. All the extracts tested negatively for anthraquinones.

Figure 4.

Phytochemical profile of the ethanolic extracts of the selected plants. 3 = abundant (heavy precipitate); 2 = fairly present (turbidity); 1 = slightly present; and 0 = absent (clear).

Total Phenolic and Flavonoid Content and Antioxidant Activity

In this study, the total phenolic content and total flavonoid content of T. aphylla and A. javanica extracts were calculated using the linear equations obtained from gallic acid (y = 9.6851x + 0.0353, R² = .9993) and quercetin (y = 31.904x + 0.0272, R² = .9996) standard curves, respectively. Their antioxidant activities were also determined at different concentrations using DPPH and H₂O₂ radical scavenging assays with ascorbic acid used as a standard antioxidant. The IC₅₀, a parameter widely used to measure the antioxidant efficiency, was calculated from the graph obtained by plotting the inhibition (%) against concentrations values and was expressed in microgram per milliliter of the extract. The results are summarized in Table 3, where a lower IC₅₀ value indicates a higher antioxidant activity. The results revealed that T. aphylla bark extract had the highest content of phenolic compounds (278.02 ± 0.16 mg GAE/100 g) followed by aerial parts extract of A. javanica (228.60 ± 2.09). The highest total flavonoids content was recorded in aerial parts extract of A. javanica (99.24 ± 0.07 mg QE/100 g). The antioxidant results showed potential free radical scavenging activity in a concentration-dependent manner. Based on the DPPH assay, the antioxidant capacity of the extracts calculated as the IC₅₀ value in µg/mL was found to be 18.39 ± 0.62 and 28.54 ± 0.53 for T. aphylla (bark) and A. javanica (aerial parts), respectively, compared with the standard antioxidant ascorbic acid (27.27 ± 0.11). However, for the H₂O₂ assay, the highest antioxidant activity (IC₅₀ = 154.17 ± 0.78) was exhibited by A. javanica aerial parts extract followed by T. aphylla bark extract (IC₅₀ = 252.94 ± 1.86) comparing with ascorbic acid (IC₅₀ = 164.90 ± 0.37).

Table 3.

Total Phenolic Content (mg GAE/100 g Extract), Total Flavonoid Content (mg QE/100g Extract) and Antioxidant Activities of the Ethanol Extracts of Tamarix aphylla and Aerva javanica by DPPH and H₂O₂ Methods With IC₅₀ (µg/mL) Values.

Antimicrobial Screening

Each plant extract, at the concentration of 100 µg/mL, was screened for antimicrobial activities. The results, graphically represented in Figure 5, showed that T. aphylla bark extract and A. javanica root extract exhibit moderate to weak activity against all the six tested human pathogens compared with the standard antibiotics (ampicillin and nystatin). However, the aerial parts extract of A. javanica showed no activity against the pathogens tested.

Figure 5.

Graphical representation of the susceptibility study (expressed as a zone of inhibition) of the extracts of A. javanica root (R) and T. aphylla bark (B) against test pathogens.

Literature-Based Proof of Biological and Pharmacological Activities

Interestingly, some medicinal uses recorded in this study were consistent with those given in the literature, for example, the use of A. javanica as a diuretic (Arbab et al., 2016), and to remove acne from the face (Perry & Metzger, 1980), and the external use of T. aphylla for wound healing and skin diseases (Akhlaq & Mohammed, 2011; Marwat et al., 2008; Shaheen et al., 2014). Some medicinal uses were instead conflicting with the recorded applications, such as the use of T. aphylla root decoction for stomach ache that was previously cited for tuberculosis, smallpox, leprosy, and contagious ailments (Ali et al., 2019). In addition, A. javanica aerial parts extract was used to treat headache and hypertension; however, in the literature, it was used to heal dysentery, gonorrhea, hyperglycemia, and cutaneous infections (Garg et al., 1980; Khan et al., 2012). The present findings concerning the medicinal use of T. aphylla parts in wound treatment have been examined previously (Adnan et al., 2015; Iqbal et al., 2017). Yusufoglu and Alqasoumi (2011), who evaluated the wound healing activity of T. aphylla extract using the excision wound model to monitor wound contraction and wound closure time, reported that the plant extract accelerated the wound healing process by reducing wound contraction and wound half closure time. The use of the bark of T. aphylla to treat hepatitis, an inflammation of the liver caused by viruses, was also reported previously (Akhlaq & Mohammed, 2011; Nawwar et al., 2009) but was not confirmed in in vivo studies; hence, the plant extract has shown antioxidant activity (Mahfoudhi, Ben Salem, et al., 2016; Qadir et al., 2014) and a significant anti-inflammatory effect when tested in Wister albino rats using a carrageenan-induced paw edema test (Al-Jaber & Allehaib, 2017; Iqbal et al., 2017). A significant proportion of the respondents reported the use of T. aphylla leaves to relieve joint pain, headache, and rheumatism. The effectiveness of T. aphylla extract as an analgesic was assessed previously by Al-Jaber and Allehaib (2017) using two methods: acetic acid-induced writhing and the hot plate method in which both the alcoholic extract of T. aphylla showed high analgesic activity. Qadir et al. (2014) performed another earlier study using Eddy’s hot plate method and demonstrated significant analgesic activities of T. aphylla. The phytochemical profile of T. aphylla bark extract, presented in Figure 4, indicated high amounts of tannins with moderate amounts of flavonoids, glycosides, and triterpenes, and the absence of alkaloids and anthraquinones. This result agrees with that recorded by Mahfoudhi, Prencipe, Mighri, and Pellati (2014) and Nawwar et al. (2009). Many active phytochemical compounds have been isolated and characterized from T. aphylla extracts (Table 2). The ethnobotanical results (Table 1) indicated that all parts of A. javanica have broad applications in folk medicine to treat various diseases. Aerial parts decoction of A. javanica is used to treat hypertension and headache and is used as a diuretic. This is possible, because diuretics are medications designed to increase the amount of water and salt expelled from the body as urine (Ellis, 2019) and are often prescribed to help treat high blood pressure, of which headache is symptom, but this use is not yet proven. Documented pharmacological activities of different parts of A. javanica, presented in Table 2, indicated the numerous biological activities of the plant. The phytochemical profile of A. javanica extracts is represented in Figure 4 and is comparable to the findings reported previously by many authors (Arbab et al., 2016; Nawaz et al., 2015). In addition, its root extract exhibited high amounts of alkaloids and saponins, whereas the aerial parts extract possessed triterpenes and flavonoids in abundance. The presence of such phytochemical constituents in these plants supports their biological activities and verifies their uses in traditional medicine to treat various diseases.

Antimicrobial Activities of the Extracts

From the present preliminary investigation, the root extract of A. javanica was found to display weak to moderate activity against all six tested pathogens (Figure 5). Similar results of the antimicrobial activity of A. javanica were reported previously (Mufti et al., 2012; Sharif et al., 2011). T. aphylla bark extract also showed weak to moderate activity against all the pathogens tested (Figure 5), and it was consistent with the findings obtained by Mahfoudhi, Ben Salem, et al. (2016). The weak antimicrobial activity of A. javanica and T. aphylla extracts suggested that the medicinal uses of these species may be attributing to biological properties other than antimicrobial properties.

Total Phenolic and Flavonoid Content and Antioxidant activity

Polyphenols are the most common and widely distributed group of secondary plant metabolites that are believed to assist in the prevention of oxidative stress and inflammatory diseases (Działo et al., 2016). The present results indicate that all the extracts tested contain a significant amount of phenolic and flavonoid content (Table 3), in agreement with the phytochemical profiles of these plants (Figure 4). The results also showed that T. aphylla and A. javanica extracts exhibited potential free radical scavenging activity in both DPPH and H₂O₂ assays in a concentration-dependent manner (Table 3). This finding likely correlates with their high total phenolic and flavonoids content because several studies implied that the antioxidant activity of plant extracts is strongly related to the polyphenol content (Ahmed et al., 2015; Kim, Chun, Kim, Moon, & Lee, 2003). The radical scavenging effect of the T. aphylla bark extract, which had a greater quantity of total phenolic compounds (278.02 ± 0.16 mg GAE/100 g), was also determined to be stronger in DPPH (IC₅₀ = 18.39 ± 0.62 µg/mL) and H₂O₂ (252.94 ± 1.86 µg/mL) assays. The total phenolic content of T. aphylla obtained in this study was lower than that reported by Mahfoudhi, Grosso, et al. (2016), whereas its antioxidant capacity was comparable to the findings obtained by Yusufoglu and Alqasoumi (2011) who evaluated the antioxidant activity of the ethanolic leaf extract of T. aphylla collected from the Al-Kharj region of Saudi Arabia. The aerial parts extract of A. javanica depicted high contents of both total phenolic (228.60 ± 2.09 mg GAE/100 g) and total flavonoids (99.24 ± 0.07 mg QE/100 g), whereas its root extract depicted moderate contents at 122.46 ± 0.12 mg GAE/100 g and 18.94 ± 1.66 mg QE/100 g for total phenolic and flavonoids, respectively. These results were higher than the values reported by Nawaz et al. (2015), who found that different extracts of A. javanica species from Pakistan contained 0.11 ± 0.01 to 3.54 ± 0.36 g GAE/100 g of total phenolic and 0.041 ± 0.001 to 2.02 ± 0.15 g QE/100 g of total flavonoids. A. javanica aerial parts extract exhibited high antioxidant activity in both DPPH (IC₅₀ = 28.54 ± 0.53 µg/mL) and H₂O₂ (IC₅₀ = 154.17 ± 0.78 µg/mL) assays compared with the standard antioxidant ascorbic acid (DPPH: IC₅₀ = 27.27 ± 0.11 µg/mL; H₂O₂: IC₅₀ = 164.90 ± 0.37 µg/mL). The present results were consistent with the findings obtained by Munir and Sarfraz (2014), but it was more potent than that achieved by Eltayeb, Eltayeb, and Salhimi (2017) who reported IC₅₀ values ranging from 46.5 ± 2.2 to 78.3 ± 3.6 µg/mL in DPPH. Accordingly, Arbab et al. (2016) reported that A. javanica extract had the highest antioxidant activity in the b-carotene-linoleic acid bleaching assay. However, T. aphylla extract showed the lowest IC₅₀ value in DPPH compared with ascorbic acid, confirming their potent antioxidant activity. Figure 6 also indicates good antioxidant activity of A. javanica extracts in both DPPH and H₂O₂ scavenging assays (low IC₅₀ values). It could be significantly correlated with its total phenolic and flavonoid contents (Table 3). Owing to its good antioxidant activity, A. javanica has been reported to be used to treat chronic and degenerative ailments that come from oxidative damage caused by free radicals (Nawaz et al., 2015). These findings of antioxidant activities enhanced the capacity of T. aphylla and A. javanica extracts in the reported traditional medicinal uses and suggest that these plants may be considered potential sources of new antioxidant drugs.

Figure 6.

Comparison of the antioxidant capacities, IC₅₀ (µg/mL) values of T. aphylla bark (B), A. javanica aerial parts (A.p), and A. javanica root (R), extracts and the standard antioxidant ascorbic acid in DPPH and H₂O₂ scavenging activities.

Implications for Conservation

The ethnobotanical information concerning medicinal plants and their health benefits is still lying unclaimed in the Asir region of Saudi Arabia. This study has documented essential information regarding the ethnopharmacological and folklore uses of two plant species used by the inhabitants of the study area to treat approximately 18 human ailments. There was strong agreement among the informants in the uses of the plants. The highest UV reported for the species indicated their importance in the traditional medicine of the area. The literature review showed that most of the therapeutic uses of these plants were similar to their medicinal uses in many other countries of the world. These findings highlight the significance of their traditional uses. The results revealed a significant yield for all classes of the biologically active phytochemicals. In addition, biological activity screening revealed a significant free radical scavenging activity. Hence, the traditional uses of these plants are likely to be due to their antioxidant potential and other unexplored biological properties. Thus, further research should be conducted on these medicinal plants to identify and isolate of compounds with antioxidant, anti-inflammatory, and analgesic activities for clinical use. This study has contributed to the documentation of indigenous knowledge on medicinal plants regarding the Asir region, one of the most affluent biodiversity areas in the Kingdom of Saudi Arabia. It has laid a significant background that can help to explore the medicinal values and create a database of medicinal plants available in Saudi Arabia, thereby contributing to the effort of conservation of these species.