Bioassay-guided Identification of Bioactive Compounds from Senna alata L. against Methicillin-resistant Staphylococcus aureus

: Senna alata (Linn) Roxb. plant is widely used to manage various infections in folkloric medicine. Methicillin-resistant Staphylococcus aureus (MRSA) infection continues to be a major global public health problem. This study aims to investigate the bioactive components of S. alata leaves active against MRSA. The leaves of S. alata were sequentially extracted and fractionated using standard methods and screened for activities against MRSA. The diethyl ether active thin layer chromatography (TLC) spot was subjected to infrared (IR) and gas chromatography-mass spectroscopic (GC-MS) studies. The aqueous extract and diethyl ether fraction of S. alata leaves elicited the highest activity against the MRSA. The GC-MS analysis of the fraction produced 15 eluates; only the sub-fraction 13 was effective. The TLC analysis of the sub-fraction 13 revealed three spots; only the sec-ond spot produced activity. The GC-MS result of the spot showed six peaks. The spectral results for peak 3 match the data from the IR study suggestive of 9-octadecenoic acid methyl ester. Senna alata leaves possess bioactive compounds closely related to 9-octadecenoic acid methyl ester with potent antibacterial activity against MRSA.


Introduction
Infectious diseases are among the top causes of global mortality, morbidity and disability [1,2]. The pathogens such as Staphylococcus aureus form a serious burden on health due to the related infections, including pneumonia, endocarditis, and sepsis [3,4]. The methicillin-resistant Staphylococcus aureus (MRSA) is one of the most critical multidrugresistant (MDR) pathogens mostly encountered in hospitals. [5][6][7]. The global incidence of MRSA infection has increased the burden on the health care system and growing death over the years. [8]. Besides, the MRSA infection still extends to the community population. [7]. Due to the frequent emergence of antimicrobial resistance, limited options are available for treating MRSA-related infections [9]. The ineffectiveness of the available antibacterial agents necessitates the search for new compounds for use against resistant bacteria. [10].
Plants have contributed to the general health and well-being of the populace for a long time [11]. About 75% of the people globally depend on traditional medicine for their basic health requirements [12]. Many therapeutic compounds employed in orthodox medicine originate from plants [12]. Plants synthesize various secondary metabolites with different chemical diversity and biological actions [13]. Plants and their secondary metabolites have been reported to serve as sources of antimicrobial agents [14][15][16][17]. For years, attention has been shifted to investigate phytochemical compounds with potential antibacterial activity, particularly against MDR bacteria [18]. Given the roles of medicinal plants in curtailing infections disease, there is a need to continue the search for novel therapeutic agents to pave the way for the discovery of new therapeutic agents [19,20].
The plant Senna alata (Linn) Roxb. synonymous with Cassia alata (Leguminosae), is commonly distributed in Asia, Brazil, Australia, and all-over African nations, including Egypt, Somalia and Nigeria [21]. The plant is a tropically erect annual herb that grows up to 2-5 meters in high. It possesses big, leathery and slippery compound leaves bilateral that usually fold at night [22]. It has a pod fruit with small and square-shaped seeds [23]. In English, this medicinally important plant is called candle bush, craw-craw, Acapulco or ringworm bush plant [21]. The local names of the plant are Asunwon oyinbo in Yoruba, Nelkhi in Igbo, and Hantsi in the Hausa languages of Nigeria [24]. The plant is used as herbal preparation to manage hepatitis, gastroenteritis, constipation, dermatitis, eczema, jaundice, diarrhea [25], and bacterial infections [26]. The plant's leaves, stems, and roots have been ingested as a decoction to treat wounds, respiratory tract, and skin infections in Northern Nigeria [25]. Also, in Cameroon, the leaves, stems and bark of S. alata were documented to be used to treat gastroenteritis and skin infections [21]. Its leaves, bark and stem are effective against intestinal parasitosis and syphilis as a decoction in China, Philippines and India [27]. Keeping in view the ethnopharmacological indication and promising therapeutic properties of S. alata for treating infectious ailments, this study was conducted to screen the leaves extracts of S. alata for activity against MRSA and identify the bioactive compounds likely responsible for the activity.

Plant collection and identification
The whole plant Senna alata was sourced from Jaji village, along Kaduna-Zaria Road, Kaduna State of Nigeria. The authentication of the plant material was conducted at the Herbarium section of the Biological Science Department, Ahmadu Bello University (ABU), Zaria, Nigeria. The herbarium specimen voucher number obtained was 2421.

Extraction and fractionation
The extraction and fractionation were conducted based on the procedures used by   [29]. The fresh leaves of the S. alata were kept to dry under shade until a uniform was attained. The dried leaves were powdered using mortar and pestle into fine particles, packaged in a tightly closed container, and kept in a cool and dry environment. The Soxhlet apparatus exhaustively extracted one kilogram (1 kg) of the dried powdered S. alata leaves with 2.5 liters of petroleum ether (60-80 grades). The marc of the petroleum ether extraction was then extracted with 2.5 liters absolute methanol with the aid of the Soxhlet apparatus. The marc of the methanolic extraction was dried and percolated with 1000 ml deionized water for 24-hours for the aqueous extraction. The extracts (petroleum ether, methanolic, and aqueous) were concentrated separately on a water bath set at 45 0C. The values for percentage yield of the extracts were calculated in relation to the formula below: One hundred grams (100 g) of the crude aqueous extract was suspended in water (500 ml) and successively partitioned with ethyl-acetate (5 × 500 ml) and diethyl ether (5 × 500 ml) to form the corresponding fractions. The ethyl-acetate and diethyl ether were evaporated to dryness while the residual aqueous was removed by placing the fraction on a water bath set at 50°C. The fractions were stored in a container separately before use. Each fraction was made into solutions with distilled water for each experiment. The percentage yield for each fraction was determined according to the formula above.

Phytochemical studies
Qualitative and quantitative phytochemical investigations were done on the crude aqueous leaves extract of S. alata to test the occurrence of phytochemical constituents in the extract according to the procedure outlined by Sofowora (1993) [30].

Test organism
The procedure previously used by   [32] was adopted. The methicillin-resistant Staphylococcus aureus (MRSA) was sub-cultured on sterile Nutrient Agar (NA) plates and incubated at 37 0C for 48-hours before each antibacterial testing.

Preparation of the test organism (MRSA)
The test organism used for the study was a standard strain of MRSA (ATCC33591) obtained from the Department of Veterinary Public Health, Ahmadu Bello University, Zaria, Kaduna, Nigeria.

Antibacterial screening of Senna alata leaves extracts and fractions against MRSA by Agar well diffusion method
The various extracts (petroleum ether, methanol and aqueous) and the aqueous fractions (ethyl acetate, diethyl ether and aqueous) of S. alata leaves were investigated for antibacterial effect against MRSA by agar well diffusion procedure as reported by   [32]. In this method, a 24-hour culture of MRSA was suspended in a sterile bottle that contained nutrient broth. Gradually, normal saline was added to obtain turbidity identical to Marcfland standard 0.5, which corresponds to about 108 cells/ml. This was then diluted to produce106 cells/ml that were used in the experiment. Then 1 ml of the test organism (106 cells/ml) was inoculated into Petri plates (90 mm diameter) for the antibacterial susceptibility test. Then wells (6 mm diameter and 4 mm deep) were punched in the agar using a sterile cork borer. The wells were bored such that not to be closer than 15 mm to the edge of the plate and enough apart to overcome overlapping the inhibition zones. Some of the wells were filled with 25 mg/ml of either one extract or fractions, while some were filled up with one of the solvents as control. An adequate time was given to allow the material to diffuse considerably into each of the media. The plates were then turned upside down and kept in an incubator at 36 0C for 48-hours. Subsequently, the various inhibition zones were observed as an index of antibacterial activity.

Determination of the zone of inhibition
Following the incubation, the plates were checked for antibacterial activities. The compounds of the discs diffused through the medium and formed a concentration gradient. Hence, the compounds of the wells formed in clear round zones of inhibition, which were measured with naked eyes using a transparent scale.

Thin-layer chromatographic analysis of diethyl ether fraction obtained from Senna alata crude aqueous leaves extract
The most active fraction (diethyl ether) of aqueous extract of the S. alata leaves was investigated for the active compound using thin-layer chromatography (TLC). The TLC analysis was done on pre-coated silica gel TLC plates (60 F254) and subsequently developed by two solvent systems of different polarities; n-butanol: acetic acid: water (6:1:2) and chloroform: acetic acid (9:1). The crude diethyl ether fraction was dissolved in methanol and spotted on the pre-coated G60 F254 TLC plates and developed in each solvent system. The various spots were detected using the following detecting agents; iodine vapor, ammonia vapor, ultraviolet (UV) light at 366 nm and UV in ammonia. The number of spots, colors, and retardation factors (Rf) values for each spot were calculated. The various spots of the crude diethyl ether fraction were investigated for activity against the MRSA by the agar overlay protocol 2.8. Antibacterial screening of various TLC spots obtained from diethyl ether fraction of Senna alata crude aqueous leaves extract against MRSA by Agar overlay method Three (3) milliliters of nutrient broth in a sterile capped tube were inoculated with MRSA. The inoculated bottle was incubated in an incubator at 37 0C for 48-hrs. The developed TLC plates were aseptically placed in sterile Petri dishes. About 15 ml of melted top NA was measured using a pipette into three (3) different sterile capped bottles and allowed to cool to about 45 0C. To each bottle containing the melted NA, about 0.8 ml of the Nutrient broth containing MRSA was pipetted and vortexed to mix the test organism. The content of each bottle; melted top agar containing-MRSA was then poured onto each of the Petri dishes containing the developed TLC plates and control plates containing the unspotted TLC plates developed in the solvent system; chloroform: acetic acid (9:1). The plates were gently tilted back and forth to have an even distribution of the test organism, then allowed to gel fully and incubated at 37 0C for 48-hours. Subsequently, the various inhibition zones were measured as an index of antibacterial activity.

Determination of the zone of inhibition
Following the incubation, the plates were checked for antibacterial activities. The compounds of the well diffused through the medium and formed a concentration gradient. Thus, the compounds of the plates resulted in a clear zone of inhibition for a particular organism and were measured by naked eyes using a transparent scale.

Column chromatographic analysis of the diethyl ether fraction obtained from the Senna alata crude aqueous leaf extract
The most active fraction (diethyl ether) was chromatographed over a silica gel column (mesh size: 70-230) and successively eluted with solvents to increase polarity using n-hexane and ethyl-acetate. The eluates were then applied onto a pre-coated TLC card of silica gel 60G254 (thickness 0.1mm) plates and eluted with methanol: chloroform (4:1). [33] The eluates with the same Rf values were pooled together. The column eluted with nhexane: ethyl acetate (40:60) afforded the most active eluate.

Gas chromatography-mass spectrometry of the active spot (Spot 2)
The TLC active spot (spot 2) was analyzed with a gas chromatography-mass spectrometry (GC-MS) (GC-MS-QP2010PLUS) analyzer. The data were obtained on an elite-1 (100% dimethyl polysiloxane) column (30 0.25 mm 1 µmdf). The carrier gas used was helium (99.99%) with a flow rate of 1ml/min in the split mode (10:1). An aliquot of 2 µl of ethanol solution of the sample was injected into the column with the injector temperature at 250ºC. The gas chromatography (GC) oven temperature was started at 110ºC and held for 2min. The temperature was elevated to 200ºC at the rate of 10ºC/min without holding. Holding was allowed at 280ºC for 9 min with a program rate of 5ºC/min. The temperature of the injector and detector were set at 250ºC and 280ºC, respectively. Ion source temperature was maintained at 200ºC. The mass spectrum of compounds in the samples was obtained by electron ionization at 70 eV, and the detector was operated in scan mode from 45-450 atomic mass units (amu). A scan interval of 0.5 seconds and fragments from 45 to 450 Da was maintained. The total running time was 36 minutes.
2.9. Infra-red spectroscopy of the active spot (spot 2) The infra-red (IR) spectroscopic analysis of the active spot was carried out on the KBr disc using Fourier transform (FT) Spectrophotometer.

Percentage yield of the Senna alata leaves extracts
The sequential extraction of one kilogram (1 kg) of the powdered Senna alata leaves with petroleum ether, methanol and water yielded 150.2 g, 104.03 g and 204.57 g representing an extractive value of 15.02%, 10.40% and 20.46%, respectively.

Percentage yield fractions per 100 g of S. alata crude aqueous leaves extract
The partitioning of 100 g of the S. alata crude aqueous extract with ethyl-acetate, diethyl ether, and the aqueous fraction yielded 1.68 g, 1.83 g, 2.45 g corresponding to 16.80%, 18.30% and 24.50% of the starting material, respectively.

Antibacterial activities of leaves extract of S. alata
The methanol and aqueous leaves extract of S. alata elicited remarkable inhibition against MRSA. However, the petroleum ether extract was not active against the bacterial isolate. The MRSA was resistant to the standard antibacterial agent used (oxacillin). The aqueous and methanol extracts exhibited the highest (14.5 mm ± 0.01) and lowest (11mm ± 0.11) zone of inhibition, respectively, against the test organism. The outcomes of the antibacterial screening of the petroleum ether, methanol and aqueous extracts of S. alata leaves against MRSA are shown in Table 1.

Antibacterial activities of fractions obtained from the crude aqueous extracts of S. alata on MRSA
The diethyl ether fraction inhibited the MRSA (14.00 ± 0.03 mm). However, the ethylacetate and aqueous fractions were ineffective against the bacterial isolate. The output of antibacterial effects of the ethyl-acetate, diethyl ether and the crude aqueous leaves extract of S. alata against MRSA are presented in Table 2.   Table 3. Table 3. Antibacterial screening of the various silica gel columns eluates of the diethyl ether fraction obtained from the crude aqueous leaves extracts of S. alata on MRSA.

Antibacterial screening of the various spots of sub-fraction 13 of the diethyl ether fraction obtained from aqueous leaves extract of S. alata on MRSA
The result of TLC analysis of the active column eluates (sub-fraction 13) revealed three (3) different spots when the plate was developed with solvent system chloroform: methanol (4:1). The antibacterial screening of the spots against the MRSA revealed that only spot 2 produced activity against the MRSA (15.00 ± 0.05 mm). The antibacterial activity of the various spot of sub-fraction 13 on the MRSA is shown in Table 4. Table 4. Antibacterial Screening of the different spots of sub-fraction 13 obtained from the diethyl ether fraction of aqueous leaves extracts of S. alata on MRSA.

Spot
Concentration (mg/ml) Zone of inhibition (mm) 1 100 0.00 ± 0.00 2 100 15.00 ± 0.05 3 100 0.00 ± 0.00 The values are the mean of two measurements across each zone of inhibition and in duplicates measured in millimeters (mm). The zero values indicate no inhibition.
3.9. Thin layer chromatographic studies of diethyl ether fraction obtained from crude aqueous leaves extract of S. alata Different colors were observed on exposing the TLC plate of the active diethyl ether sub-fraction (fraction 13) of the crude aqueous leaves extract of S. alata to different detecting mediums. Under direct light, bright yellow (Spot 1) was observed. When this plate was exposed to ammonia vapor, two spots were observed: yellow (Spot 1) and pink (Spot 2). Nine yellow Spots were observed when the TLC plate was exposed to iodine vapor. These spots changed to various colors when observed under UV light in the presence and absence of ammonia vapor. Viewing the plate under UV light, in the absence of ammonia revealed 9 variedly colored spots: spot 1 purple (Rf=96), spot 2 blue (Rf=94), spot 3 light purple (Rf=80), spot 4 orange (Rf=73), spot 5 purple (Rf=66), spot 6 blue-green (Rf=45), spot 7 light purple (Rf=38), spot 8 purple (Rf=28) and spot 9 purple-green (Rf=19). However, when the plate was exposed to UV light, in the presence of ammonia, spot 1 changed from purple color to blue color, spot 3 changed to pink color, while spots 2, 4, 5, 6, 7, 8, 9 appeared as orange, blue-green, green, light purple, light purple, light blue and green respectively.

Rf values and color of fraction 13 of diethyl ether fraction obtained from crude aqueous leaves extract of S. alata when viewed under different detecting medium
Thin-layer chromatographic analysis of fraction13 of the diethyl ether showed three spots with Rf values of 45, 54, and 93. All three spots turned yellow with iodine vapor. Spot 2 turned pink with ammonia vapor and appeared yellow under UV light (366 nm), while spot 3 turned light blue under the UV light, as shown in Table 5. 3.11. Infra-red spectroscopic analysis of TLC spot 2 of the active column eluate of fraction 13 obtained from S. alata diethyl ether column eluate The FT-IR spectrum of TLC Spot 2 from sub-fraction 13 of diethyl ether column eluates was obtained as KBr disk and shown in figure 1, which contains seven (7) vibrational bands assigned to their respective functional groups as shown in Table 10

Discussion
Plant-derived products play a crucial role in exploring active biomolecules to treat infectious diseases, currently causing serious health challenges globally, especially in developing nations [34]. Therefore, it is essential to intensify research on natural products to establish the secondary metabolites responsible for activity against infectious diseases [35,36]. The current study elucidates the antibacterial potentials of various extracts and bioactive components of the aqueous leaves extract of Senna alata against Methicillin-resistant Staphylococcus aureus (MRSA). A compound related to 9-octadecenoic acid methyl ester was found to have potent antibacterial activity against MRSA.
Based on the current investigation, the aqueous extract of the plant produced the highest extractive and could contain the highest amount of secondary metabolites present in the plant. Our findings are in line with the work of Saito et al. (2012) [38], where the 100g of milled S. alata leaves produced 25.4% aqueous extract. However, the work of   [40] has shown that the methanol extract produced the highest yield.
Interestingly, this investigation has shown that the crude aqueous extract of S. alata  [25], [43], [38], [42] demonstrated the inhibitory effect of aqueous extract of S. alata against S. aureus, which corroborates with the outcome of this study. On the contrary, a previous report by   [40] has shown that the S. aureus was resistant to aqueous leaf extract of S. alata. In addition, the antibacterial activity elicited by the methanol extract of S. alata against the MRSA in this work agrees with the findings of   [45] and   [47]. The difference in the antibacterial activities produced by the methanol and aqueous extracts of S. alata leaves in the current work might be related to the secondary metabolites varying degrees of solubility in categories of solvents based on the polarity, which also corroborates with the higher extraction yield in the aqueous extract [48].
Bioassay-guided fractionation is an effective procedure to discover novel therapeutic agents via obtaining active fractions or isolated bioactive agents. In this method, each fraction is usually investigated for biological activity, and subsequently, the most active portions are eventually fractionated for further evaluation [49]. The chemical contents and potential mechanisms of activity of bioactive compounds usually vary in different parts of the plants, as well as a difference in the solubility of the secondary metabolites in different solvents [50]. In this study, the crude aqueous leaves extract of S. alata afforded ethyl-acetate and diethyl ether fractions. However, only the diethyl ether fraction elicited an antibacterial activity related to the higher amount of the bioactive agents in the fraction, as shown by its higher extractive value than the ethyl-acetate fraction.
Phytochemical determination gives an overview of the possible category of the plants' secondary metabolites and their quantity in a particular fraction which could guide the isolation strategy of the bioactive components. [51] Based on this research, the aqueous extract of S. alata leaves revealed saponins, alkaloids, emodin, tannins, steroids, anthraquinones, and flavonoids that could be associated with the antibacterial effects. The outcome agrees with the previous research by El-Mahmood & Doughari (2008) and   [54]. Generally, the anti-infective action of plants is related to the chemical compounds including tannins, phenols, saponins, steroids, alkaloids, flavonoids and many other compounds via various processes [55]. Polyphenol agents such as tannins, flavonoids, and alkaloids possess antimicrobial effects [56,57]. Flavonoids, including quercetin and kaempferol have antimicrobial actions by inhibiting the action of the bacterial enzymes [55,58,59]. The antimicrobial action of flavonoids is also associated `with their capability to produce a complex with soluble, extracellular proteins as well as bacterial cell walls, whereas tannins inhibit microbial adhesions, transport proteins, and enzymes [60,61]. Besides, flavonoids destabilize microbial cell membranes [62]. Therefore, these phytochemical compounds in S. alata could be responsible for its antimicrobial activities.
The observation in the current work that only sub-fraction 13 of the column fractions of the diethyl ether fraction of the crude aqueous S. alata leaves extract showed activity concurs with the work of   [40], in which only one of the column fractions elicited antibacterial activity. Furthermore, a single spot was observed to possess bioactivity following the active diethyl ether column fraction to silica gel TLC analysis. The bioactive spot (Spot 2) of the sub-fraction 13 obtained from the diethyl ether fraction of the crude aqueous leaves extract of S. alata being the only spot effective against the test organism possesses a hydroxyl group (-OH) in its structure as shown in its IR spectrum assignable to the vibrational band at 3320 cm-1, which is broad and of low intensity. This is typical of the -OH group found in fatty acids, and it belongs to -OH bonded to a carboxyl group carbon atom. The presence of the carboxyl group in the structure of spot 2 of the sub-fraction 13 has been confirmed by the GC-MS spectral data. The loss of two oxygen atoms (2O) from the fragmentation of the molecular ion at m/z 296 to form the fragment ion peak at m/z 264 indicated that this loss of oxygen atom emanates from a carboxyl group, which is typical of fatty acids methyl ester [63].
The FT-IR spectrum of spot 2 of sub-fraction 13 also showed a carbon-carbon double bond (C=C) due to the stretching vibration band at 1684 cm-1 (Table 10). The presence of this bond was confirmed in the GC-MS spectral data because the fragmentation of the fragment ion at m/z 137 is accompanied by a loss of carbon-carbon double bond (C=C) to produce the fragment ion at m/z 112. The orientation of the C=C bond is cis configuration. This is confirmed by the presence of C-H out-of-plane bending vibration (670 cm-1) of an olefinic bond since the bending vibration at 950-970 cm-1 (characteristic of trans configuration) is absent in the IR spectrum of spot 2 of the sub-fraction 13. Furthermore, the C-H bending vibration at 1514 cm-1 results from the presence of a terminal methyl (CH3) group in the eluate. The GC-MS spectral data confirm this because the fragment ion at m/z 83 loses a methyl group and one hydrogen atom to form the fragment ion at m/z 69. This is a diagnostic of unsaturated fatty acid with a terminal methyl group [64].
In this work, the IR spectra data of spot 2 of the sub-fraction 13 agrees with that of cis-Octadecenoic acid methyl ester [65]. In addition, the entire fragment ions in the GC-MS spectra data of spot 2 of the active sub-fraction13, including the molecular ion and base peak ion, are in line with the data reported for methyl 9-octadecenoate by   [67]. Therefore, based on the data presented in this work and the literature, and by comparing with the library search result, spot 2 of the active sub-fraction 13 was determined to be 9-octadecenoic acid methyl ester (E). Besides, the molecular formula of the active spot 2 of the sub-fraction13 was deduced as (C19H35O2) based on the information obtained from its molecular ion at m/z 296; this was supported by the findings of  [69] that the oleic acid was 75.03 of total essential oil of S. alata leaf extract using GC-MS. Besides,   [71] have shown that oleic acids are among the major fatty acids present in S. alata, confirming the presence of fatty acids in the S. alata leaves in the current work. Many researchers, including   [73],   [75] as well as   [77], have previously used Fourier transform infrared (FT-IR) spectroscopy and GC-MS to analyze the various phytocompounds.
The antibacterial action of the active spot 2, which seems to be a fatty acid methyl ester based on the library search and spectral analysis, is in line with the findings of Igwe & Onwu (2015) [69], who reported the antibacterial effects of essential oil from S. alata leaves extract against S. aureus. Similar reports by   [79],   [81],   [83] and   [85] have shown that longchain unsaturated fatty acids, including oleic acids found naturally are effective bactericidal against pathogens including MRSA. In addition,  [87] in their work reported the effectiveness of unsaturated fatty acid esters and unsaturated fatty acids esters derivatives against S. aureus and MRSA. However, the precise mechanism of antibacterial action is precisely unknown. These reports support the observation made on the antibacterial activity of the identified compound against MRSA in the present study, which could explain the traditional applications of S. alata in treating bacterial infections.

Conclusions
The Senna alata leaves possess varying degrees of antibacterial activities against MRSA. The antimicrobial activity of the plant against MRSA was likely due to the 9-Octadecenoic acid methyl ester (E) component of the plant. In addition, 9-Octadecenoic acid methyl ester (E) could serve as a lead compound for further development to discover novel therapeutic agents as an alternative treatment for drug-resistant bacterial infections. Therefore, we recommend further spectroscopic studies like a proton (3H) and Nuclear magnetic resonance (NMR) to ascertain the structure of the identified compound. Also, pharmacological and toxicity studies on the identified phytochemical compound should be conducted to establish the molecular basis of its antibacterial action and safety profile.  Data Availability Statement: All data related to the manuscript are presented in the manuscript. The raw data can be obtained from the corresponding author based on reasonable request.