Hibiscus Sabdariffa as Nuclear Stain for Cytological Buccal and Cervical Smear

Introduction: Sudan is one of the biggest countries that produces and exports hibiscus Sabdariffa in Africa. The possibility of HS staining cytological specimens is not well explored. Therefore, the present study aimed to evaluate the staining quality of Hibiscus Sabdariffa on cytological buccal and cervical smear as a cheap viable nuclear stain alternative to hematoxylin. Methods : This study was an experimental descriptive study conducted at Alfajr College for Science and Technology from (June to September 2022). A total of 30 smears, 15 buccal smears collected from students and 15 cervical smears were retrieved from Alfajr's histopathology lab. Results: All (30) slides showed good staining quality with 20% concentration Hibiscus Sabdariffa water extract when mordanted with Iron, and acidified with acetic acid. Conclusion: Hibiscus Sabdariffa is good as nuclear staining for a buccal and cervical pap smear and can replace hematoxylin by adjustment of concentration, time and pH.


INTRODUCTION
Hibiscus Sabdariffa (HS) is a plant belonging to the Malvaceae family which is a vascular flowering plant, popularly known as (Roselle) or Red Sorrel in English and composed of over a hundred species (Ananthalakshmi et al., 2016;Joshua et al., 2021;Konar et al., 2019;Saxena et al., 2021).The colour of calyces varies from white-yellow to dark red due to anthocyanin (El-Hashash et al., 2022).Hibiscus sabdariffa is cultivated in many countries around the world and Sudan is one of these countries and known as (Karkade) (Abd-alhafeez et al., 2014; Kuşçulu, 2021).Hibiscus Sabdariffa plants contain anthocyanin, organic acids, proteins, flavonoids, vitamins, and polysaccharides (S.Benard et al., 2015;N Mahadevan, 2009), and thus have a variety of industrial, nutritional, and medical uses (Agbede et al., 2017).Hibiscus is used to lower blood pressure, stimulate diuretic effects, and reduce high cholesterol, cosmetics, and food colourants.Anthocyanins are responsible for colour and extract from different plant materials and Hibiscus Sabdariffa is the most important one (S. A. Benard et al., 2015;El-Hashash et al., 2022;Pattanittum et al., 2021).HS extract has a colour ranging from red, and purple to blue, and the optimum pH for staining range from 2.5 to 4.0 (Solomon A. Benard, 2008).The deep red colour of Hibiscus Sabdariffa extract is soluble in water and convert into pink by the addition of HCL, and bluish to green by alkali (Maaza, 2014).
Sudan is one of the biggest countries that produces and exports hibiscus Sabdariffa in Africa (Abd-alhafeez et al., 2014).Synthetic dyes, hematoxylin of them are well known to expose laboratory workers to adverse health effect as allergic and toxicity (Hartika et al., 2021), moreover the shortage of hematoxylin reported by several studies (Dapson et al., 2010).Therefore, is necessary to find a natural, safe, less costly and harmful, and easy-to-obtain stain.In the current literature, several studies reported the possibility of Hibiscus Sabdariffa staining histological tissue sections, parasites, and fungi (Omorodion & Achukwu, 2017; Saxena

Sample Collection:
Samples were collected from clinically healthy buccal mucosa.The participants were asked to rinse their mouths using tap water twice.The buccal surface was scraped using a spatula, the material was smeared on glass slides and fixed with 95% ethyl alcohol for 15 minutes.

Staining Method:
Cervical and buccal smears were brought in water, followed by hibiscus extract for 30 minutes, followed by bluing in running tap water for 3minute, followed by orange G-6 stain for 3 minutes, followed by 10 dips in 95% Ethanol, then eosin Azure for 3 minutes.Then all smears were rinsed in 70%, 95%, and 100% Ethanol for 2 minutes, cleared in xylene for 2 minutes and mounted with a permanent mounting medium (DPX).

Slide examination:
The staining quality of Hibiscus Sabdariffa was examined by two observers.The slides were evaluated according to microscopic criteria of the nuclear stain, cytoplasm stain, stain intensity, and contrast.Each one of the criteria was given a score of 0 or 1.According to the sum of the scoring, the slide was then graded into 4 grades; 0= poor, 1= satisfied, 2= good and 3= excellent.Poor is referred to as the absence of stain on the nucleus/cytoplasm, and no nuclear details/ difficult assessment on the cytoplasm.Satisfy is referred to the presence a pale of stain on the nucleus/ cytoplasm and no nuclear details/ difficult assessment of cytoplasm.Good is referred to the presence of stain on the nucleus/cytoplasm comparable to hematoxylin, and the presence of nuclear details but chromatin not clearly defined/ difficult assessment on the cytoplasm.Excellent is referred to the presence of stain on the nucleus comparable to hematoxylin, and the presence of nuclear details clearly defined (Zaimy, 2017).

RESULTS
Hibiscus Sabdariffa solution stained the nucleus of superficial and intermediate cells dark violet, whereas the cytoplasm of superficial stained with pink and intermediate with blue.The granules of neutrophils in the cervical smear stained blue-black with pink cytoplasm, and Red blood cells stained red.All (30) slides showed good staining quality with 20% concentration Hibiscus Sabdariffa water extract when mordanted with Iron, and acidified with acetic acid.).We noticed that when using a 25% concentration of HS, the extract solidified and this is probably due to pectin, a previous study reported a such finding (Hashim, 2006).Moreover, the nucleus stained blue-black with running tap water for 10 minutes (bluing), whereas stained red-purple with water washing for 1 minute as well as hematoxylin, such findings reported by (Alshamar & Dapson, 2021a).In the present study HS failed to stain the bar body and this result comparable to (Omorodion & Achukwu, 2017).In contrast, a previous study done by (Saxena et al., 2021) on buccal smear to evaluate Hibiscus as a counter stain for hematoxylin comparable to Pap stain, found that Hibiscus gave the poorest result and failed to stain the cytoplasm when ammonia was used to alkalify the pH.Several previous studies focused on HS as a substitution for hematoxylin