Herbal remedies used for the management of urolithiasis in Abbottabad, Northern Pakistan

Ghulam Mujtaba Shah1a, Nasir Shad2a, Muhammad Sajid3, Asma1, Abid Naeem4*, Fahim Ullah Khan3, Nadia Jabeen3, Mazna Urooj1, Said Muhammad1 & Muhammad Hasnain5 1Department of Botany, Hazara University Mansehra, Pakistan 2Jiangxi Provincial Key Laboratory of Silviculture, College of Forestry, Jiangxi Agricultural University, Nanchang 330045, China 3Department of Agricultural, Hazara University Mansehra, Pakistan 4Key Laboratory of Modern Preparation of Traditional Chinese Medicine, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, 1688 Meiling Road, Nanchang 330006, PR China 5State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of Environment, Northeast Normal University, Changchun 130117, China aThese authors contributed equally to this article *Email: anaeemktk@gmail.com


Introduction
Urolithiasis or kidney stone formation is the third leading urinary system disease affecting 10-15% of the population worldwide. Generally, it emanates from tiny crystals, which later results in stone, and subsequently grows further and accumulates on the interior surfaces of the kidney (1). Kidney stones or calculi are named based on the identified locations in the urinary system, such as renal calculi, urinary tract stone disease, urinary calculi, urolithiasis, nephrolithiasis and ureterolithiasis. Urinary stones can be removed/expelled via urine if smaller in size through the ureter from the kidney (2). It is observed that sometimes the stones may not pass through the ureter, which produces severe pain if not managed properly, resulting in serious medical consequences like extreme obstruction, infection, haemorrhage and hydronephrosis in the urinary system.
The major health problems faced by the people of Pakistan are linked to the kidney, and recently more than 1 million cases of kidney stones were reported (3). It is estimated that 15 % of all the people in Pakistan will suffer from urolithiasis at some point in life. Men tend to be affected more frequently than women (4). Moreover, urolithiasis is the most common illness amongst the people of Northern Pakistan. Different treatment options are available to remove calculi, such as surgical removal, lithotripsy and laparoscopy. Despite the higher treatment costs, these techniques may produce a risk of causing acute renal injury, which decreases renal function and also recurrence of calculi observed in some cases with a possible recurrence rate of 10%, 33% and 50% at 1st, 5th and 10th year respectively (5). Costly diagnostic and treatment procedures for the therapeutic management of patients require better adequate alternative therapies. Phytochemicals are responsible for the therapeutic activity of plant species and can treat various ailments and possess potential antiinflammatory, antibacterial, analgesic, diuretic, antioxidant and antifungal properties. Natural products derived from medicinal plants, either as pure compounds or as extracts, provide opportunities for developing new drug leads due to the unmatched chemical diversity and lower cost compared to their synthetic chemical counter-parts (6). Due to the recent surge in demands for chemical diversity in screening programmes, search for therapeutic drugs of herbal origin increased due to their attractive properties worldwide. Botanicals and herbal preparations for medicinal purposes contain different types of bioactive compounds like flavonoids, alkaloids, saponins, tannins, sterols and other organic and inorganic constituents. Some medicinal plants and marketed herbal preparations are reported to be effective in treating and preventing kidney stones recurrence with fewer side effects (7). In recent times, the discovery of antilithiatic drugs from natural products has gained a lot of importance.
The practiced medicinal plants by traditional healers and locals for the cure of kidney stones possess various bioactive chemical compounds, and some of the chemical constituents from different plant species have been used in anti-urolithiatic formulations (6). Various phytoconstituents have been isolated from traditionally used medicinal plants and have been found to be effective in preventing kidney disease/stones (6,8). Diet from natural food (vegetative plants and fruits) also plays a vital role in either preventing or promoting the formation of kidney stones, depending on the types of nutrients and chemical constituents found in the plants (6). Those plants which contain phytoconstituents like (flavonoids, alkaloids, tannins, saponins, phenolic compounds, organic and inorganic constituents, plant proteins etc.) are responsible for anti-urolithiatic activity (9). The World Health Organization (WHO) stated that almost 4 billion (80%) people worldwide take advantage of herbal remedies for their healthcare needs. Furthermore, the remaining 25% of conventional drugs marketed in developed countries are mostly derived from natural products such as vinblastine, quinine, reserpine and aspirin (10). It is estimated that about 50000-70.000 plant species are utilized for medicinal purposes by traditional and modern healthcare systems worldwide (11). Pakistan has different climatic and ecological zones enriched with floral diversity, containing almost 6000 species of important flowering plants, including 500 species of extreme therapeutic importance. Some of these plants are used in the locally practiced medical system (Hikmat) (12).
A thorough literature search revealed that a lot of work had been done on the medicinal plants in the Abbottabad region (13)(14)(15)(16)(17)(18)(19)(20). However, yet no report is available regarding kidney diseases. Therefore, it would be important to document these medicinal plants employed for treating kidney stones. The current study will not only be helpful in the conservation of resources but also helps in preserving the fast-disappearing traditional knowledge and may provide a base for pharmacological research.

Materials and Methods
This study was authorized by the Department of Botany and Office of Research, Innovation and Commercialization of Hazara University (ORIC-HU), Mansehra, Pakistan. Informed consent was obtained from each informant before conducting the interview process.

Study area
The Abbottabad district covers an area of 1967 km 2 and falls between 34° 9 -34° 21 North latitude and ʹ ʹ 73° 10 -73° 13 East longitude, predominantly ʹ ʹ mountainous terrain (Fig. 1). The average peak elevation ranges from 2500-2700 m and the Miranjani peak (3313 m) is the highest point in the district. These peaks dominate the landscape and are a part of the lesser Himalayas. The Dongagali peaks unite with the right banks of the Jhelum and Kunhar river from the north. It includes various offshoots that differ in altitudes from 2500-2700 m. Several other peaks also project around this range, especially from the west, with 600-1500 m of range. The overall population of Abbottabad is around 1169432 and the urban population is estimated to be 1519751. The topography of Abbottabad is mainly composed of gullies and steep slopes, with metamorphic rocks formed of limestone. Recently, frequent deforestation and pasturage triggered soil erosion, which has made the parent rock shallower and silty loess (21). The average temperature in the region ranges from 34.3-3.4 °C, with occasional snow. Most rainfall occurs in winter and monsoon; the average being recorded is around 1200 mm per year. The major tribes of the region include Gujjar, Awan, Karlal, Syed, Jadoon, Abbasi, Rajput, Kashmiri and Mughal. Most of the population depends on forests and agriculture (primary occupation), although cattle grazing occurs near the adjacent hilly areas. Nevertheless, some people are also involved in local labour and trade. The most commonly grown crops in the area include rice, maize, wheat, tobacco etc. Recently, due to increased illegal tree cuttings, extensive fires, illegal settlements, urban encroachment and growing pollution, the area is under severe threat (22).  (Table 1). Questions asked to the informants about the medicinally used plants were mainly related to the use of the plant, its local name, habitat, place and time of collection, used parts and categories, fruit and vegetable consumption, way of drug preparation and administration and at last, diseases treated. Plant collection was done during the blooming and fruiting stage, approved by local inhabitants to verify the authenticity of the collected plant. After drying, the specimens were pressed, poisoned and finally mounted on herbarium sheets. Interviews were conducted mostly in fields; otherwise, photographs were shown for identification. Females were interviewed indirectly through male family members. Participants were briefed about the research objectives and were allowed to discontinue the interview at any time. The native language (Urdu and Hindko) has been used as a medium of communication. Later, an English language questionary proforma was filled for each informant.

Identification of plants
The collected plant species were identified with the available Flora of Pakistan, and their names were identified through the literature search and key plant-related websites such as the plant list (www.theplantlist.org), international plant name index (http://www.ipni.org) and GRIN taxonomy site (http://www.ars-grin.gov/cgi-bin/npgs/html/queries.p l). For voucher specimens, standard herbarium techniques (23,24) were strictly followed and were deposited in the Hazara University Herbarium, Pakistan for future references.

Quantitative and correlations analysis of ethnomedicinal data
The obtained data were analyzed and presented using quantitative indices, descriptive statistics and correlation analysis for all the collected plant species.

Relative Frequency Citation (RFC)
Relative frequency citation was calculated while ignoring the use-categories following the formula (25).
) RFC shows the importance of each species in the study area given by the FC (FC is the number of local informants reported the uses of the species) divided by the total number of informants (N).

Use value (UV)
Use value (UV) determines the importance of each plant species utilized by locals. It was calculated using the following formula (26).
Where "UV" indicates the use-value of individual species, "Ui" is the number of uses recoded for a given species by each informant, and "N" represents the total number of informants in the study. Fidelity level percentage (FL%) Fidelity level (FL) is the percentage of plant specimens that the interviewees claim to be used for the same particular purpose. Fidelity level shows the importance of each species for a specific disease. Its value can be calculated for common diseases as follows: Np is the number of individuals claiming to utilize a group of plant specimens to cure a specific disease, and N is the number of people utilizing herbal remedies to treat any illness (23,27,28).

Results and Discussion
The study found that 38 species belonging to 37 genera and 27 families are utilized to treat different urinary tract infections (  (9), like Asparagus racemosus Willd.
(root, 5-7 g, powder) used by Siddha for the treatments of urolithiasis (29,30). A large number of preclinical studies support the traditional uses of medicinal plants to prevent kidney stones (31), such as an in vitro experiment (32) found that the aqueous extract of Bryophyllum pinnatum leaves was effective in dissolving calcium phosphate. Another study (29) used the ethanolic extract of Asparagus racemosus on albino rats and found that the concentration of calcium and phosphate was significantly reduced. In short, most of the modern drugs have been originated from the traditional knowledge of medicinal plants by identifying/isolating/modifying the specific chemical constituents for specific diseases and then performing its clinical trials to evaluate their safety and efficacy and also the effective dosage and its route of administration.

Species richness for the treatment of urolithiasis
A total of 38 species from 37 genera belonging to 27 families were documented, which were used by local people for the treatment of urolithiasis in the study area. Asteraceae family (5 species) was the most frequently mentioned among plant families, followed by Amaranthaceae, Liliaceae, Saxifragaceae, Apiaceae, Euphorbiaceae, Polygonaceae and Urticaceae (2 species each) and the other plant families were recorded with only one medicinal plant species each (Fig. 2). Most of the registered plant species in the study area have been practiced by the Ayurveda Pharmacopoeia of India, TCM, Siddha, Unani for the urolithiasis treatment (2,9); some of them have been tested in preclinical studies and some of the claims were scientifically proved for the same diseases as in the therapy/practice.

Ethnomedicinal utilization of plant parts for the treatment of urolithiasis
A total of eight different plant parts were used for the management of urolithiasis by the local community (Fig. 3). Leaves were the most widely used part (28.94%), followed by the whole plant (21.05%), rhizomes and roots (13.5%), flowers and seeds (7.89%) and fruits and stem (2.63%). Because of the lower level of communication means, ignorance, poverty and the absence of modern therapeutic facilities, the people of the rural areas still depend on traditional medicines for curing various ailments (Table 1). Plant parts and the way of their utilization have a significant role in preventing any disease. It has been found that some of the traditionally used plant parts contain phytoconstituents which can be used as antiurolithiasis agents, such as Bergenia ciliata leaves (ethyl acetate extracts) (33) and Bryophyllum pinnatum leaves extracts (32). In a study, the pulp extracts of Citrullus lanatus were found to be effective against urolithiasis, while the seed extracts did not show any effectiveness (34). In contrast, the seeds of Citrullus lanatus were used traditionally against urolithiasis. However, in this study, most of the reported species based on the part of utilization and their purpose against urolithiasis matched with the phytoconstituents used in pre-clinical stages. Some species were found with no pharmacognostic study records, therefore, recommended for further screening.

Preparations of herbal remedies
The local communities used the native diversity of medicinal plants for treating urolithiasis disease by following several administrational ways; decoction was the popular and dominant way of preparation (52.6%), followed by powder (18.4%), extracts (15.7%), juice (7.8%) and cooked (5.2%) (Fig. 4).
Mostly used medicinal plants demonstrate that approximately 650 species are utilized as mono-or polyherbal remedies and are used in the form of decoction and acts as diuretic substances (31). The preparation of polyherbal remedies is considered less toxic, safe and effective by traditional medicinal systems and is inexpensive than allopathic therapy (2). In traditional therapies, plant parts used, their preparation and the route of administration may have a significant role in the pharmacological action of medicinally used plant taxa.

Important ethnomedicinal species
The UV of plant species determines the relative importance of plants in the study area.   Use value and RFC value are dynamic as it changes with the area and depends on the local people's traditional knowledge, so the UV and RFC value may vary within the same area or area to area and community to community (35). Correlation analysis between RFC and UV was significant at the 0.01 level (0.803**), indicating that the local people have significant traditional knowledge of the cited species (Table 3).
Fidelity Level percentage shows the importance of each species for specific urolithiasis disease in this study ( Table 2). Those species with above 50 FL% in the study area were considered for further literature search, parts used, pharmacological properties and chemical constituents against urolithiasis. The similarity of traditionally used/reported species was also evaluated against the phytoconstituents assessed in preclinical models of urolithiasis (Table 4). An invitro experiment (33) found that phenolic compounds (ethyl acetate extracts) from the Bergenia ciliata leaves effectively dissolved calcium phosphate and oxalate stones, where cystone was more effective than phenolic compound against urolithiasis. The leaf extracts of Bryophyllum pinnatum and their chemical constituents are useful for preventing kidney stones formation and decreasing urinary calcium levels.
The current reported medicinal plants activities against urolithiasis may be due to the presence of bioactive compounds in the plant parts used traditionally and some of these parts of the plants have been investigated for their bioactive compounds against urolithiasis, while some are still unknown. Previous studies found the effectiveness of the chemical constituents against urolithiasis derived from plant parts that were practiced in the present study, such as Citrullus lanatus and Tribulus terrestris (seed) (34,36), Equisetum ramosissimum, Oxalis corniculata and Phyllanthus niruri (whole plant) (37)(38)(39), Zea mays (stem) (40), Asparagus racemosus, Agrimonia eupatoria, Polygonum aviculare, Trichodesma indicum, Xanthium strumarium and Trianthema portulacastrum (leaves) (29, 41-44), Rumex acetosa (root) (45), Celtis australis, Tamarindus indica, Ficus carica and Citrus sinensis (fruit) (46)(47)(48), Dioscorea deltoidea and Cyperus rotundus (rhizome) (49,50). The present study demonstrated that the chemical compounds from plant parts in the modern health systems and their utilization (51) may have been practiced earlier by the ancient traditional medicine systems in their own way and may have been transferred from generation after generation.
Previous studies demonstrated that Bergenia stracheyi had been widely used by traditional Indian, Nepalese and Chinese medicine systems for the treatment of bladder and kidney stones and several other diseases (52)(53)(54). Similarly, the present study recorded Bergenia stracheyi with cent fidelity level, used against kidney stones that release stones from the kidney, yet no medical therapy has been performed. Another plant species Flemingia grahamiana was continuously reported in the study area, but it has not been well explored as antiurolithiasis in pre-clinical trials. Therefore, further clinical trials are required, especially of these two species, to record their efficacy against urolithiasis.

Cultural significance
The WHO promotes and advises the documentation of traditionally used medicinal plants for the treatment of various diseases around the world (55). Various clinical trials have been conducted to evaluate the efficacy and safety of various traditional medicinal plants. About 75% of drugs currently used directly or indirectly came from traditional medicinal plants (56). Listing of high value ethnomedicinal plants is essential and has a significant value in determining human-plant relationships (57). It is important to involve the local communities in developing useful drugs plants that can socioeconomically uplift them. The present study found that only older people and traditional healers have more knowledge of these useful medicinal plants, which may transfer generation after generation. Hence, proper documentation of ethnomedicinal knowledge of valuable medicinal plants in the country is necessary and should be analyzed using modern research techniques to find useful compounds and molecules, and then should be further evaluated in the preclinical and clinical models for the treatment of various diseases. There is a dire need to find better alternative drugs due to the adverse effects of conventional medicines and herbal medicine can be a possible solution for that purpose only after passing through vigorous chemical and biological screening procedures (58). It is also important that new crops are introduced to ethnobotanists and anthropologists, who can gather and document the traditional knowledge and significance of those plants, which will help to conserve the biodiversity and lead to sustainable utilization of these useful plant resources. The currently collected information will help to drive the research work forward and can help in the scientific evaluation of the safety and efficacy of the traditional herbal remedies used by the tribes, especially the less familiar remedies (59,60). Consequently, people can be informed and guided about the scientifically proven efficacious drug treatments for various diseases, which will improve their health conditions.

Status of medicinal plants of Abbottabad region
The primary sources of medicinal plants in Pakistan are rangelands and forests, mainly utilized as traditional medicines (74)(75)(76). The biodiversity of medicinal plants in the region is under serious threat because of anthropogenic activities, overgrazing, deforestation, timber smuggling, ruthless and unscientific collection (22). These anthropogenic activities lead to the alteration in habitat, fragmentation and severe habitat destruction, which resulted in significant habitat loss. Furthermore, climate change and plant invasions are the other serious threats in this regard (77,78). Both in situ and ex situ conservation measures are required to conserve the regional flora, such as massive reforestation, natural gas supply, community awareness, providing energy efficient cookstoves, and establishing botanical gardens and proper guiding training of medicinal-plant collectors, which may help in flora conservation. However, these efforts will only get results if they are cross-sectoral and promote cooperation and collaboration among the natural resource managers, government agencies, traditional medical practitioners and various other interest groups.

Conclusion
The current study reported that the local communities widely practice indigenous herbalbased medicines and its utilization remains to be a viable and better alternative therapeutic option for the rural and underprivileged sections of the communities. However, precise traditional knowledge about medicinal plants was found among few people (mostly older people) in the local communities. Therefore, proper exploration, documentation and preservation of traditional ethnomedicinal knowledge are required before it disappears forever and needs to be preserved for future generations. Our survey revealed that there are still some claims that warrant experimental investigations, implying that a lot can still be learned from exploring medicinal plants in the region, which may lead to finding valuable phytoconstituents in plants that are effective against urolithiasis and also other diseases. All these ethnomedicinal plants require a thorough scientific investigation for isolation, identification, biochemical assays, toxicities and evaluation of pharmacological activities of the phytoconstituents, especially of the high-fidelity level plants. This study will help in preserving the useful and immensely important knowledge of traditionally used medicinal plants in the region and promote the importance of ancient healing practices in the coming generations. It also emphasizes the need for a holistic approach to engaging the local communities in the conservation of medicinal plants by sustained harvest and cultivation in their natural habitat due to the immense ecological and pharmacological importance.