The Aqueous Stem Bark Extract of Alstonia boonei Exhibits Anticataract Activity in Sprague Dawley Rat

In Africa, Alstonia boonei is used folklorically for the management of the multitude of conditions including cataract, which accounts for 50% of cases of blindness in the region. The current study set out to probe the traditional use of the aqueous extract of Alstonia boonei stem bark (ABE) as an anticataract remedy using Sprague Dawley rat models. We investigated the probable phytochemical constituents in the extract, in vitro antioxidant potential, and its in vitro aldose reductase inhibition. For the anticataract investigations, diabetic cataract was induced using galactose in 3-week-old Sprague Dawley rats, and age-related cataract was induced by the administration of sodium selenite to 10-day-old rat pups. Cataract scores in both models were determined after treatment with 30, 100, and 300 mgkg−1 doses of ABE and 10 mlkg−1 of distilled water. Lens glutathione, total lens protein, soluble lens proteins (alpha-A) crystallin, and aquaporin 0 levels in the enucleated lens homogenates were determined. Changes in lens to body weight were also determined with histopathological analysis done on the lenses in the selenite-induced cataract model. The presence of alkaloids, tannins, flavonoids, glycosides, and triterpenoids was identified in the extract. The extract inhibited aldose reductase activity with IC50 of 92.30 μgml−1. The 30, 100, and 300 mgkg−1ABE-treated rats recorded significantly (p < 0.05) reduced cataract scores indicating a delay in cataractogenesis in galactose-induced cataract and in selenite-induced cataractogenesis as well. Markers of lens transparency such as AQP0, alpha-A crystallin, and total lens proteins and lens glutathione levels were significantly (p < 0.05) preserved. In conclusion, this study establishes the anticataract potential of the aqueous stem bark extract of Alstonia boonei in Sprague Dawley rat models.


Introduction
Cataracts are described as lens clouding that results in visual impairment [1,2]. In Africa, it accounts for about 50% of cases of blindness [3,4] and 54.8% of the cases in Ghana [5]. Cataract can be congenital and age-related or may be a result of underlining diseases such as diabetes and is therefore characterised as a disease of priority.
Age-related cataract remains a leading cause of blindness worldwide and is believed to increase the risk of death [6]. Multiple mechanisms are involved in the development of diabetic cataract. Earlier studies have identifed the lens' response to hyperglycaemic environment through processes such as increased oxidative stress [7] which plays a major part in the pathogenesis of diabetic cataract.
To eliminate cataract, the WHO launched the Vision 2020 campaign [1], which culminated in the realisation that individuals who patronised cataract surgery were much less than expected, i.e., 523 out of 2000 patients [8]. Tis means that signifcant proportions of people sufering from cataracts do not seek medical help or resort to alternative treatment options other than surgery [9,10]. As such, the efectiveness and relevance of these alternative treatment options in the management of cataracts need to be investigated.
One of the known alternatives to surgery largely employed in cataract treatment in Africa is the use of plant and plant-based products [11]. One such plant extensively used in the management of cataract in Africa is Alstonia boonei although there has not been any scientifc justifcation of its use in that regard.
Alstonia boonei De Wild (Apocynaceae) is a large tree commonly found in the dry lowlands and rainforests across West Africa. Te stem bark has a myriad of uses in folklore medicine, including its use as antimalarial and antivenom [12] and anticataract [13]. Te current study, therefore, set out to investigate the anticataract activity of aqueous extract of the stem bark of Alstonia boonei in galactose-and seleniteinduced cataracts in Sprague Dawley rats and provide scientifc evidence to its traditional use.

Plant Collection. Alstonia boonei stem bark was collected from Asakraka-Kwahu in the Eastern Region of Ghana in
November 2020 and validated at the Department of Herbal Medicine, KNUST (Voucher specimen No: KNUST/HMI/ 2022/SB002). Te plant material was then sun-dried.

Animals.
Sprague Dawley rats (3 weeks old and 10 days old) of both sexes were procured from the Center for Plant Medicine Research (CPMR), Mampong, Eastern Region, Ghana, and housed in stainless steel cages at the Department of Pharmacology Animal Housing Facility, UCC. Rats were allowed free access to commercial chow and water. Animals were humanely handled throughout the entire duration of the studies.

Extraction.
About 640 g of the stem bark of Alstonia boonei was blended using a heavy-duty blender (37BL85 (240CB6) Waring Commercial, USA). Te fne powder obtained was transferred into a percolator and macerated with 4.5 L of distilled water for 3 days. Te mixture was fltered, and the fltrate was concentrated at 40°C using a rotary evaporator (Rotavapor R-210, BUCHI, Switzerland) and further concentrated at 50°C using an industrial oven (Gallenkamp OMT Oven, SANYO, Japan). A brownish solid is produced which was stored at 4°C, reconstituted using PBS when needed, and referred to as Alstonia boonei extract (ABE) [14].

Preliminary Phytochemical
Screening. Te phytochemical contents of ABE were studied using methods as described by Harborne [15].
2.6. In Vitro Antioxidant Assays 2.6.1. Total Flavonoid Content. Te method described by Chang et al. [16] was used. Briefy, a mixture of 0.5 ml of the extract in 0.3 ml of 5% NaNO 2 and 0.3 ml of 10% AlCl 3 was prepared. Te mixture was incubated at 25°C for 30 min in a laboratory incubator (Yohmai IN-601, Stains, France), 2 ml of 1 moll −1 NaOH was added, and absorbance was determined at 415 nm using a spectrophotometer (BioTek-800-TS absorbance reader, Agilent, Santa Clara, USA). A standard curve was established with quercetin, and the total favonoid capacity of the extract was extrapolated from the curve.

Total Phenolic Content.
Te Folin-Ciocalteu method as described by Hudz et al. [17] was used in this determination.
A 0.6 ml mixture containing 0.1 ml of 0.5 N Folin-Ciocalteu and 0.5 ml of ABE was prepared and incubated at 25°C for an hour, and 2.5 ml of 2% NaHCO 3 was added afterwards. Te resulting solution was further incubated at ambient temperature for 90 min, and the absorbance was determined at 760 nm using a spectrophotometer (BioTek-800-TS absorbance reader, Agilent, Santa Clara, USA).

Total Antioxidant Capacity.
Total antioxidant capacity estimated as gallic acid equivalent (GAE) was determined as earlier described by Urbano et al. [18]. A fnal mixture containing 1 ml of ABE and 3 ml of a solution made up of sulphuric acid, disodium phosphate, and ammonium molybdate at concentrations 0.6 M, 28 mM, and 4 mM, respectively, was incubated (Yohmai IN-601, Stains, France) at 95°C for 90 min. Te absorbance of the mixture was determined at 695 nm (BioTek-800-TS absorbance reader, Agilent, Santa Clara, USA) upon cooling. Gallic acid was used to establish a standard curve with the GAE of ABE being extrapolated from the obtained curve.

Free
Radical Scavenging Activity. Te method described by Sharma and Bhat [19] was used in this determination. Briefy, 1 ml of varying concentrations of ABE (i.e., 2000−62.5 μgml −1 in methanol) was added to a 20 mgl −1 mixture of methanol and DPPH. Te resulting solution was incubated (Yohmai IN-601, Stains, France) under shade for 30 min at room temperature, and the absorbance was determined afterwards at 517 nm (BioTek-800-TS absorbance reader, Agilent, Santa Clara, USA), using ascorbic acid (i.e., 100−0.78 μgml −1 ) as the positive control. Te IC 50 , the concentration at which there was a 50% decrease in the absorbance of DPPH, was then determined using the relation: %DPPH scavenging ability � absorbance of control − absorbance of ABE absorbance of control x 100%. (1)

Aldose Reductase Inhibition Assay.
In this assay, unrefned aldose reductase (AR) in enzyme solution was collected as the supernatant from a homogenized rat lens which was centrifuged (Bioswisstec Hermle Z 36HK, Schafhausen, Switzerland) at 5000 × g for 30 min at 4°C [20]. Varying concentrations of quercetin (control) and ABE (10-1000 μgml −1 ) were prepared as inhibitors of AR activity using phosphate-bufered saline (PBS). To 50 μl of the enzyme solution in a cuvette, 50 μl ABE, 50 μl 0.1 M phosphate bufer (pH 7), and 50 μl 0.03 mM NADPH were added. Te enzyme reaction was initiated with the addition of 50 μl Dxylose to the mixtures, and absorbance was determined at 340 nm after 180 s using BioTek 800 TS absorbance reader (Agilent, Santa Clara, USA). Te inhibition of AR activity was expressed as a percentage of quercetin [21].
2.9. Galactose-Induced Cataractogenesis. Te method used by Kyei et al. [22] and modifed by Amanfo et al. [23] was employed in this study. Briefy, 3-week-old Sprague Dawley rats had their lenses examined for cataractogenesis using the slit lamp guided by the graded score and put into fve groups (n � 5) and treated as indicated below for 6 weeks: Group 1 received only 10 mlkg −1 distilled water p.o. for 6 weeks; Group 2 received 3000 mgkg −1 galactose p.o. daily plus 10 mlkg −1 distilled water p.o. for 6 weeks; and Groups 3-5 received 3000 mgkg −1 galactose p.o. daily plus 30, 100, or 300 mgkg −1 ABE p.o. for 6 weeks, respectively. Te lenses of all rats used in the study were examined for degree of opaqueness weekly using a Macro II-B Slit Lamp (Marco-Lombart Instrument, Japan) and scored based on the scale detailed (Table 1) as described by Amanfo et al. [23].

Blood Sugar Determination.
Blood samples were taken, before the start of treatment and weekly, from the rat tail vein. All samples were taken after an overnight fast, and fasting blood sugar levels were determined using a glucometer (Accu Chek Performa, Roche Diagnostics, USA).

Lens-to-Body Weight Ratio Determination.
Rats were weighed before the study, and the lens was also weighed when extracted at the end of the study. Te lens-to-body weight ratio was then computed.

Lens Glutathione (GSH) Assay.
Te extracted lenses were homogenized in PBS and centrifuged (Bioswisstec Hermle Z 36HK, Schafhausen, Switzerland) at 5000 × g for 30 min to obtain a supernatant used for subsequent determinations. Te supernatant obtained was used to determine GSH levels in the lens using a glutathione ELISA kit (Shanghai, China) in accordance with the guidelines provided by the manufacturer. Te measurements were made in duplicate.

Total Lens Protein
Assay. Using a bicinchoninic acid (BCA) ELISA kit purchased from Shanghai Chemical Ltd, China, we determined the lens total protein from the supernatant using the manufacturer's guidelines. Te measurements were made in duplicate.
2.14. Selenite-Induced Cataractogenesis. We employed the model detailed by Kyei et al. [24] and modifed by Amanfo et al. [23] in this investigation. Te 10-day-old rats were given subcutaneous shots of 15 μmolkg −1 sodium selenite daily for two days and put into fve groups (n � 4), and treatments meted out are detailed as follows: Group 1 received only 10 mlkg −1 distilled water p.o. and 12 hourly for 21 days without any sodium selenite being given to the rats; Group 2 received only 10 mlkg −1 distilled water p.o. 30 min before the sodium selenite challenge and 12 hourly for 21 days; and Groups 3-5 received 30, 100, and 300 mgkg −1 ABE p.o. 30 min before the sodium selenite challenge and 12 hourly for 21 days, respectively.
Te pupils of the pups were dilated via ophthalmic tropicamide installation on day 22, and lenses were accessed for cataractogenesis using the slit lamp.

Cataract
Grading. Te extent of cataract was graded as described by Sippel [25] and is detailed in Table 2.

Lens Soluble Proteins (CRYAA) and
Aquaporin 0 (AQP0) Levels. Rat lenses were extracted at the end of the study and homogenized in PBS. Te resultant solution was centrifuged (Bioswisstec Hermle Z 36HK, Schafhausen, Switzerland) at 5000 × g for 30 min, and the supernatant was collected for CRYAA and AQP0 assays using ELISA kits (Shanghai Scientifca Chemical Ltd, China) in accordance with the manufacturer's guidelines. Te measurements were all done in duplicate, and the experiment was repeated three times.

Statistical Analysis.
Results were plotted and analyzed using GraphPad Prism for Windows 8.0.1 (GraphPad Software Inc., USA). Values were considered statistically signifcant at p < 0.05.

In Vitro Antioxidant Assay.
Tere was an observable increase in the total antioxidant capacity of all standards used with increased concentrations of ABE (Figures 1(a)-1(c)). Te extract was realized to exhibit a dose-dependent scavenging activity (Figure 1(d)), and the standards of 725.43, 663.49, and 534.85 μgg −1 were realized for vitamin C, gallic acid, and quercetin, respectively.

Aldose Reductase Inhibition Assay.
In this assay, ABE was realized to show an inhibition of aldose reductase activity with an IC 50 of 92.30 μgml −1 (Table 4).

Efect of ABE on Blood Sugar Levels.
From the timecourse curve, the normal control (NC) rats were observed to show a steady level of blood galactose over the 5-week period. Upon galactose administration to rats, there was a steady rise in blood galactose level in the rats in the negative control (NeC) group. Treatment with ABE resulted in a signifcant (p < 0.05) drop in galactose levels at all dose levels studied over the 5-week period (Figure 2(a)).
Te area under the time-course curve (AUC) confrmed the observed blood galactose levels of 26.32 ± 0.55 in NC rats which increased to 72.59 ± 4.996 upon galactose administration in NeC rats. Tis rise in blood sugar was signifcantly reduced by ABE administration at doses of 30,100, and 300 mgkg −1 to levels of 33.85 ± 2.85, 33.62 ± 0.20, and 37.95 ± 3.30, respectively (Figure 2(b)).

Efect of ABE on Galactose-Induced Cataractogenesis.
From the time-course curves obtained, galactose administration to rats caused an increase in cataract score which peaked after 1 week. However, ABE administration caused practically undetectable signs of cataractogenesis comparable with determinations made in NC rats (Figure 3(a)).
When the AUCs were determined, it established cataract score values spanning from 0 to 2 for ABE-treated rats as compared to about 13 in NeC rats (Figure 3(b)).

Efect of ABE on Lens-to-Body Weight Ratio.
Te lensto-body weight ratio was realized to signifcant (p < 0.05) increase in control rats exposed to galactose as compared to the distilled water-treated rats. Upon ABE treatment, rats recorded a signifcant (p ≤ 0.001) reduction in the lensto-body weight ratio at 100 and 300 mgkg −1 doses administered ( Figure 4).

Efect of ABE on Selenite-Induced Cataractogenesis.
Upon slit lamp analysis, it became apparent that NC rats developed no cataract over the duration of the study. 6 out of 8 eyes of rats in NeC showed grade IV cataract with the other 2 showing grade III nuclear cataract. Upon treatment with ABE, grade I cataract was only observed in 2 eyes in Peri-nuclear area opacity of lens with intense nuclear cataract 4 Total lens opacity Clear lens with less than 3 vacuoles 2 Clear lens with more than 3 vacuoles 3 Vacuoles covering entire lens surface 4 Incomplete opacity of lens 5 Complete lens opacity 4 Scientifca 30 mgkg −1 treated rats, 0 cataract recorded in rats treated with 100 mgkg −1 of extract, and 3 eyes in 300 mgkg −1 treated rats showed grade I cataract ( Figure 6).

Histopathological Analysis.
Te histopathology of NC rats presented a regular arrangement of lens fbres in the cortex (yellow arrow) (Figure 8(a)). In NeC rats with fullblown cataract, there were observable signs of lens epithelial erosion and abnormal changes in lens fbre morphology (red arrows). Again, NeC rats exhibited clear signs of distorted   lens fbres impregnated with fragmentations (Figure 8(b)).
In rats treated with ABE, there were no clear signs of disturbance to the lens fbre integrity and architecture (white arrow) (Figures 8(c)-8(e)).

Discussion
Current understanding of the precise mechanism involved in the progression of diabetic cataract is still limited, leaving signifcant gaps in our knowledge. As a result, the development of reliable preventive and therapeutic medications for this condition is yet to be achieved. Nevertheless, the galactose cataract model replicates the occurrence of secondary cataract. Compared to glucose-induced cataract, galactose-induced cataract is efcient. Te model is economical and can rapidly replicate the pathophysiology of diabetic cataract. Oftentimes, galactosemic cataract is used to investigate the mechanisms of action of drugs employed in the management of diabetes-associated complications. Moreover, galactosemic cataract develops quickly and is reversible compared to other models of diabetic cataract, such as streptozotocin and alloxan [27]. Galactosemic cataract mimics diabetic cataracts and is characterised by the detection of trace amounts of sugar in peripheral blood following the administration of galactose. Tese traces of sugar are instrumental to cataract development. On the other hand, selenite shots in the selenite cataract model, a replicate of age-related cataract, cause cataractogenesis by increasing oxidative stress due to increased levels of reactive oxygen species [28] and reduced solubility of soluble proteins such as α, β-c crystallin [29] and distortion of calcium balance [30].
In the galactose cataract model, increased galactose consumption results in hyperglycemia which in a cascade reaction leads to upregulation of aldose reductase [31,32], the enzyme involved in the polyol pathway. Aldose reductase causes galactose to be metabolized into galactitol [33]. Galactitol accumulates in the lens because it cannot cross the lens membranes by passive difusion, leading to the increased osmotic pressure of the lens and lens swelling and an increase in weight [34]. In addition, there is an increase in the expression of reactive oxygen species that can also destroy the lens through oxidative stress [35].
In contrast to the low afnity for glucose, aldose reductase has a higher afnity for galactose. Additionally, galactitol, the alcohol metabolite of galactose, has been shown to be much more challenging for sorbitol dehydrogenase to metabolize compared to sorbitol. Tus, galactosemia is more likely to cause severe cataracts in shorter time periods [36,37]. With all these disparities, the extract was able to signifcantly decrease sugar levels, effectively preventing hyperglycaemia and development of cataracts.  Figure 5: Efect of Alstonia boonei aqueous extract on lens GSH and total protein levels of Sprague Dawley rats with galactose-induced cataract. * * p < 0.05; * * p < 0.01, * * * p < 0.001; * * * * p < 0.0001, signifcance between the negative control (NeC) group and the other treatment groups (ANOVA followed by Dunnett's post hoc test). Te values plotted are the mean ± SEM (n � 5).
Moreover, Alstonia boonei extract decreased cataract scores in rats. Tis observed efect can be attributed to the extract's inhibitory efect on aldose reductase, as well as the ability of the extract to prevent potential oxidative stress caused by the build-up of ROS when galactitol accumulates in the lens. In addition, this assertion is supported by the absence of physical signs of cataract, including an increase in lens weight and loss of body weight [23]. However, a limitation of this fnding was our inability to estimate galactitol levels in addition to the measurement of blood glucose levels.
Among the various antioxidants present in the lens, the tripeptide glutathione is the most abundant and plays a crucial role in the preservation of lens transparency [38]. Furthermore, a decrease in total lens proteins is a recognized sign of precataractous changes [22]. Given this, the extract's ability to increase both glutathione and lens protein levels contributed to the improvement in lens transparency observed in the extract-treated rats.
In addition to the Alstonia boonei extract's inhibition of galactose-induced cataract, treatment with the extract also suppressed selenite-induced cataract in rat pups. Tis model serves as a valuable model for studying senile cataracts. It ofers several advantages, including the ability to rapidly and consistently induce cataract formation within a short timeframe [39]. Due to similarities observed in increased levels of calcium and insoluble proteins, reduced levels of glutathione, and vesicular formation, this model is well suited for investigating the fundamental mechanisms that underpin human cataract formation [40]. Signs of sodium selenite-induced cataracts include m-calpain activation, which results in increased calcium levels through a cascade of biochemical processes. Additionally, there is an elevation in insoluble proteins, oxidative stress, and a reduction in lens soluble proteins such as α, β-c crystallin [41]. Te preventive and therapeutic efects of phytoconstituents in age-related ocular disorders, particularly cataracts, have been extensively documented [42]. Flavonoids have demonstrated protective efects against opacifcation of the lens through inhibition of glycoxidation [43,44]. Likewise, alkaloids have been found to counteract oxidative damage induced by ROS like hydrogen peroxide [45]. Te presence of these phytoconstituents in the extract may contribute to its ability to prevent cataract formation.
Additionally, the extract demonstrated a preventive or delaying efect on selenite-induced cataracts in rodent pups. Tis outcome holds clinical signifcance as delaying the onset of cataract formation can help prevent the associated visual impairment that can impact the independence of afected individuals [46,47]. In fact, even a relatively short delay in cataract development can signifcantly improve the quality of life, reduce reliance on others, and prolong survival by about 10 years [48]. Te observed delay seen in the low-and high-dose treatment has the potential to signifcantly improve the lives of individuals, particularly the elderly, over a considerable period of time.
Te impact of oxidative stress on selenite-induced cataracts is evident from the rapid emergence of nuclear cataracts within a maximum of 5 days following sodium selenite administration. Secondary metabolites from plants such as favonoids and tannins possess antioxidant properties that enable them to eliminate ROS [49]. Te presence of these phytochemicals in Alstonia boonei extract could explain its preventive efect on senile cataract development.
Senile cataracts involve a cascade of biochemical processes, including a general reduction in protein levels and the insolubilization of soluble proteins within the eye lens [50]. Proteins serve as essential transport channels, and in the case of selenite-induced cataracts, the transport system within the lens is compromised [51,52]. Proteins such as alpha-A crystallin function as a molecular chaperone in the lens [53][54][55], whereas others such as aquaporin 0 (AQP0), also known as main intrinsic polypeptide (MIP), serve as a water channel [56]. Treatment with the extract maintained the levels of these markers within the lens, which likely contributed to the observed anticataract efect.

Conclusions
Te aqueous stem bark extract of Alstonia boonei delays and prevents galactose and selenite-induced cataractogenesis in Sprague Dawley rats and pups, respectively. Te observed anticataract activity may be attributed to the extract's ability to decrease oxidative stress in the lens. Te current study gives scientifc evidence to its claim as an anticataract agent. Future studies may, however, identify the specifc phytochemical(s) accounting for the probable anticataract efect of the aqueous stem bark extract of Alstonia boonei.

Data Availability
Te datasets generated and/or analyzed during the current study are available from the corresponding authors upon reasonable request.

Ethical Approval
Te ethical clearance for this study was obtained from the Animal Research Ethics Committee of Kwame Nkrumah University of Science and Technology for the work using rodents (reference KNUST 0019).